Magnesium-induced inner membrane aggregation in heart mitochondria: prevention and reversal by carboxyatractyloside and bongkrekic acid

Mg(2+) at an optimal concentration of 2mM (ph 6.5) induces large increases (up to 30 percent) in the optical density of bovine heart mitochondria incubated under conditions of low ionic strength (< approx. 0.01). The increases are associated with aggregation (sticking together) of the inner membranes and are little affected by changes in the energy status of the mitochondria. Virtually all of a number of other polyvalent cations tested and Ag(+) induce increases in mitochondrial optical density similar to those induced by Mg(2+), their approximate order of concentration effectiveness in respect to Mg(2+) being: La(3+) > Pb(2+) = Cu(2+) > Cd(2+) > Zn(2+) > Ag(+) > Mn(2+) > Ca(2+) > Mg(2+). With the exception of Mg(2+), all of these cations appear to induce swelling of the mitochondria concomitant with inner membrane aggregation. The inhibitors of the adenine nucleotide transport reaction carboxyatratyloside and bongkrekic acid are capable of preventing and reversing Mg(2+)-induced aggregation at the same low concentration required for complete inhibition of phosphorylating respiration, suggesting that they inhibit the aggregation by binding to the adenine nucleotide carrier. The findings are interpreted to indicate (a) that the inner mitochondrial membrane is normally prevented from aggregating by virtue of its net negative outer surface change, (b) that the cations induce the membrane to aggregate by binding at its outer surface, decreasing the net negative charge, and (c) that carboxyatractyloside and bongkrekic acid inhibit the aggregation by binding to the outer surface of the membrane, increasing the net negative charge.     

Scaling-up the delivery of dog vaccination campaigns against rabies in Tanzania

An increasing number of countries are committing to meet the global target to eliminate human deaths from dog-mediated rabies by 2030. Mass dog vaccination is central to this strategy. To interrupt rabies transmission from dogs to humans, the World Health Organization recommends that vaccination campaigns should be carried out every year in all dog-owning communities vaccinating 70% of their susceptible dogs. Monitoring and evaluation of dog vaccination campaigns are needed to measure progress towards elimination. In this study, we measured the delivery performance of large-scale vaccination campaigns implemented in 25 districts in south-east Tanzania from 2010 until 2017. We used regression modelling to infer the factors associated with, and potentially influencing the successful delivery of vaccination campaigns. During 2010–2017, five rounds of vaccination campaigns were carried out, vaccinating in total 349,513 dogs in 2,066 administrative vaccination units (rural villages or urban wards). Progressively more dogs were vaccinated over the successive campaigns. The campaigns did not reach all vaccination units each year, with only 16–28% of districts achieving 100% campaign completeness (where all units were vaccinated). During 2013–2017 when vaccination coverage was monitored, approximately 20% of vaccination units achieved the recommended 70% coverage, with average coverage around 50%. Campaigns were also not completed at annual intervals, with the longest interval between campaigns being 27 months. Our analysis revealed that districts with higher budgets generally achieved higher completeness, with a twofold difference in district budget increasing the odds of a vaccination unit being reached by a campaign by slightly more than twofold (OR: 2.29; 95% CI: 1.69–3.09). However, higher budgets did not necessarily result in higher coverage within vaccination units that were reached. We recommend national programs regularly monitor and evaluate the performance of their vaccination campaigns, so as to identify factors hindering their effective delivery and to guide remedial action.     

Thiol/disulfide interconversion in bovine lens aldose reductase induced by intermediates of glutathione turnover

The effectiveness of cysteine and cysteinylglycine to act as protein thiolating agents was investigated using bovine lens aldose reductase (ALR2) as the protein target. Disulfides of both thiol compounds appear to be very effective as ALR2 thiolating agents. Cysteine- and CysGly-modified ALR2 forms (Cys-ALR2 and CysGly-ALR2, respectively) are characterized by the presence of a mixed disulfide bond involving Cys298, as demonstrated by a combined electrospray mass spectrometry and Edman degradation approach. Both Cys-ALR2 and CysGly-ALR2 essentially retain the ability to reduce glyceraldehyde but lose the susceptibility to inhibition by Sorbinil and other ALR2 inhibitors. Cys-ALR2 and CysGly-ALR2 are easily reduced back to the native enzyme form by dithiothreitol and GSH treatment; on the contrary, Cys and 2-mercaptoethanol appear to act as protein trans-thiolating agents, rather than reducing agents. The treatment at 37 degrees C of both Cys-ALR2 and CysGly-ALR2, unlikely what observed for glutathionyl-modified ALR2 (GS-ALR2), promotes the generation of an intramolecular disulfide bond between Cys298 and Cys303 residues. A rationale for the special susceptibility of Cys-ALR2 and CysGly-ALR2, as compared to GS-ALR2, to the thermally induced intramolecular rearrangement is given on the basis of a molecular dynamic and energy minimization approach. A pathway of thiol/disulfide interconversion for bovine lens ALR2 induced, in oxidative conditions, by physiological thiol compounds is proposed.     

Modulation of aldose reductase activity through S-thiolation by physiological thiols

The glutathionyl-modified aldose reductase (GS-ALR2) is unique, among different S-thiolated enzyme forms, in that it displays a lower specific activity than the native enzyme (ALR2). Specific interactions of the bound glutathionyl moiety (GS) with the ALR2 active site, were predicted by a low perturbative molecular modelling approach. The outcoming GS allocation, involving interactions with residues relevant for catalysis and substrate allocation, explains the rationale behind the observed differences in the activity between GS-ALR2 and other thiol-modified enzyme forms. The reversible S-glutathionylation of ALR2 observed in cultured intact bovine lens undergoing an oxidative/non oxidative treatment cycle is discussed in terms of the potential of ALR2/GS-ALR2 inter-conversion as a response to oxidative stress conditions.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Unprecedented olefin-dependent histidine-kinase inhibitory of zerumbone ring-opening material

Zerumbone ring-opening derivative, 4 (10E/10Z=3/2), inhibited autophosphorylation of the essential histidine-kinase YycG existing in Bacillus subtilis constituting a two-component system (TCS). Generation of 4E-form could be regulated chemically using the difference from the ring-opening reactivity of the precursor forming of 4 and pure 4E was isolated. The stereoisomer, 4E, showed main inhibition activity of autophosphorylation of YycG (IC(50)=63.5 microM).     

Physiological thiols as promoters of glutathione oxidation and modifying agents in protein S-thiolation.

Glutathione is one of the most relevant antioxidants present in cells. It exerts its scavenging action through the involvement of efficient and ubiquitous enzymes. GSH on the other hand, because of its chemical features, can scavenge reactive oxygen species without the involvement of enzymatic systems. The study deals with the mobilization of GSH pool in a nonenzymatic antioxidant system by other physiological thiols (i.e., cysteine and cysteinyl-glycine), which are far more sensitive than GSH to oxidative conditions. These thiol compounds, in the presence of iron/EDTA, can promote oxygen activation through their oxidation to disulfides. GSH, through trans-thiolation reactions, can regenerate Cys and CysGly, which can then recycle, thus inducing a massive GSH oxidation. In these conditions, making use of bovine lens aldose reductase as a protein model, evidence is given that Cys and CysGly promote specific protein S-thiolation reactions. The possibility that GSH may be recruited in controlling cellular oxygen tension is considered.     

Using the number of koilocytes to predict HIV serostatus in women with HPV-associated SIL

OBJECTIVE: To investigate the relationship between specific cytopathologic changes, koilocyte counts and human papillomavirus (HPV) types in HIV-positive and -negative women. STUDY DESIGN: A cohort of 459 women (266 HIV+ and 193 HIV-), were examined in a multicentric study (Early Diagnosis of Neoplasia in AIDS) involving 14 gynecologic centers. Altogether, 97 women had cervical smears consistent with squamous intraepithelial lesions (SIL). Koilocytes were found in 60/97 SIL slides, subjected to quantitative counting in 30 predetermined fields. HPV genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. RESULTS: SIL lesions were four times more frequent (29%) in HIV-positive women than in HIV-negative women (10%) (odds ratio = 3.80). HPV DNA was equally frequent in both groups. There was a strong association between the number of koilocytes and HIV serostatus in both high grade and low grade SIL diagnoses. The presence of eight or more koilocytes had a specificity of 93% and sensitivity of 76% toward the diagnosis of HIV-positive status. No HIV-negative woman had a count > 8 koilocytes. No association was shown between koilocyte count and HPV genotype. CONCLUSION: An elevated number of koilocytes could suggest the possibility of HIV infection. Pap smear examination might give the first clue to HIV positivity in otherwise-unsuspected cases.