The concentration and control of cytoplasmic free inorganic pyrophosphate in rat liver in vivo

The concentration of cytoplasmic free pyrophosphate was calculated in freeze-clamped livers of rats from the measured concentration of reactants and K(eq.) of the UDP-glucose pyrophosphorylase reaction (UDP-alpha-d-glucose 1-phosphate uridylyltransferase, EC 2.7.7.9). The K(eq.) of the UDP-glucose pyrophosphorylase reaction was redetermined at 38 degrees C, pH7.0, I=0.25mol/l and free [Mg(2+)]=1mm, and was 4.55 in the direction of glucose 1-phosphate formation. The activity of UDP-glucose pyrophosphorylase in rat liver was between 46 and 58mumol of glucose 1-phosphate formed/min per g fresh wt. in the four dietary conditions studied. A fluorimetric assay with enzymic cycling was developed for the measurement of glucose 1-phosphate in HClO(4) extracts of rat liver. The calculated free cytoplasmic PP(i) concentration in nmol/g fresh wt. of liver was 2.3+/-0.3 in starved, 3.8+/-0.4 in fed, 4.9+/-0.6 in meal-fed and 5.2+/-0.4 in sucrose-re-fed animals. These values agree well with recently determined direct measurements of total PP(i) in rat liver and suggest that there is not a large amount of bound or metabolically inert PP(i) in rat liver. The cytoplasmic [ATP]/[AMP][PP(i)] ratio is 10(3) times the cytoplasmic [ATP]/[ADP][P(i)] ratio and varies differently with dietary state. The reaction PP(i)+H(2)O-->2P(i) catalysed by inorganic pyrophosphatase (EC 3.6.1.1) does not attain near-equilibrium in vivo. PP(i) should be considered as one of the group of small inorganic ions which is metabolically active and capable of exerting a controlling function in a number of important metabolic reactions.     

Changes in the concentrations of hepatic metabolites on administration of dihydroxyacetone or glycerol to starved rats and their relationship to the control of ketogenesis

1. Glycerol and dihydroxyacetone, both antiketogenic and readily metabolized, but differing in their effects on the redox state of the hepatic NAD couples, were given to starved rats and the contents of metabolites were measured in freezeclamped liver and in the blood. The object was to study the effects of changes in the redox state and of the availability of oxidizable substrates on the rate of ketone-body formation. 2. Intramuscular administration of dihydroxyacetone, glycerol or glucose to starved rats decreased the concentrations of acetoacetate and 3-hydroxybutyrate in the blood by 70-80% within 60min., whereas there was no major change in the free fatty acid concentration. 3. Dihydroxyacetone, but not glucose or glycerol, caused an immediate and sustained twofold increase in the blood lactate concentration. 4. Dihydroxyacetone and glycerol caused a rapid fall in the hepatic concentrations of ketone bodies, dihydroxyacetone being more effective. 5. This decrease was not accompanied by significant changes in the concentrations of acetyl-CoA, long-chain acyl-CoA or free CoA. 6. The hepatic glycerophosphate concentration rose about 40-fold on administration of glycerol, whereas with dihydroxyacetone the increase was only about 50%. The large increase in glycerophosphate concentration after administration of glycerol was completely prevented by pretreatment of the rats with tri-iodothyronine. Triiodothyronine-treated rats showed the same decrease in ketone-body concentrations after administration of glycerol as the untreated rats. 7. Glycerol and dihydroxyacetone caused an increase in the hepatic lactate concentration; the pyruvate concentration rose only after injection of dihydroxyacetone. 8. Both compounds increased liver glycogen. 9. Calculation of the [free NAD(+)]/[free NADH] ratios indicated that dihydroxyacetone increased the ratio in cytoplasm and mitochondria, whereas glycerol caused a prompt fall in both compartments, followed at 10min. by a slight rise in the mitochondrial compartment. 10. Dihydroxyacetone did not alter the hepatic content of ATP. 11. The findings suggest that the main reason for the antiketogenic effect of glycerol and dihydroxyacetone was a consequence of their ready metabolism and the provision of an increased supply of C(3) intermediates for conversion into oxaloacetate. Under the test conditions, neither the hepatic content of alpha-glycerophosphate nor the redox state of the NAD couples appeared to play a major role in the regulation of ketogenesis.     

Changing Mortality Profile among HIV-Infected Patients in Rio de Janeiro,Brazil: Shifting from AIDS to Non-AIDS Related Conditions in the HAART Era

Introduction

We describe temporal trends in the mortality rates and factors associated with AIDS and non-AIDS related mortality at the Evandro Chagas Clinical Research Institute (IPEC), Oswaldo Cruz Foundation (FIOCRUZ).

Methods

Adult patients enrolling from 1986 through 2009 with a minimum follow up of 60 days were included. Vital status was exhaustively checked using patients’ medical charts, through active contact with individuals and family members and by linkage with the Rio de Janeiro Mortality database using a previously validated algorithm. The CoDe protocol was used to establish the cause of death. Extended Cox proportional hazards models were used for multivariate modeling.

Results

A total of 3530 individuals met the inclusion criteria, out of which 868 (24.6%) deceased; median follow up per patient was 3.9 years (interquartile range 1.7–9.2 years). The dramatic decrease in the overall mortality rates was driven by AIDS-related causes that decreased from 9.19 deaths/100PYs n 1986–1991 to 1.35/100PYs in 2007–2009. Non-AIDS related mortality rates remained stable overtime, at around 1 death/100PYs. Immunodeficiency significantly increased the hazard of both AIDS-related and non-AIDS-related causes of death, while HAART use was strongly associated with a lower hazard of death from either cause.

Conclusions

Our results confirm the remarkable decrease in AIDS-related mortality as the HIV epidemic evolved and alerts to the conditions not traditionally related to HIV/AIDS which are now becoming more frequent, needing careful monitoring.     

Thermal niche overlap of the corner recluse spider Loxosceles laeta (Araneae; Sicariidae) and its possible predator,the spitting spider Scytodes globula (Scytodidae)

Loxoscelism is a health problem caused by the bite of spiders of the genus Loxosceles. In Chile all cases are attributable to Loxosceles laeta. It has been suggested that the spitting spider Scytodes globula may be a predator of L. laeta and control its population, which is only possible if they share the microhabitat. This study compared the thermal preferences and tolerances of the two species. Later, spiders acclimated to 15 °C and 25 °C were exposed to decreasing and increasing temperatures to determine the lower and upper critical temperatures. The preferred temperatures were lower during the morning, but there were no differences between the species. The thermal niche breadths were similar for the species, with a large overlap. Both species showed tolerance to extreme temperatures, but L. laeta showed greater tolerance to low temperatures. Both species showed acclimation of the lower critical temperatures to changes in acclimation temperatures. The similarity of preferred and tolerated temperatures was partly an expected fact, since the species share the same macrohabitat; these spider species are very common in domestic environments of central Chile. However, the results imply that their microhabitat choices are also very similar, indicating a high probability of meeting and predation, which could have important consequences in loxoscelism epidemiology.     

Dose-response effect of chlorhexidine on a multispecies oral biofilm formed on pure titanium and on a titanium-zirconium alloy

Abstract

This study aimed to test the dose-response effect of chlorhexidine on multispecies biofilms formed on commercially pure titanium (cpTi) and titanium-zirconium (TiZr) alloy. Biofilms were formed on cpTi and TiZr discs and treated two times per day with five different chlorhexidine concentrations (0.12, 0.20, 0.50, 1, 2%). The biofilms were collected for microbiological, biochemical and microscopic analyses. The significance of differences among groups was evaluated by linear regression, ANOVA, Bonferroni and Tukey tests. The mean number of colony-forming units decreased as the chlorhexidine concentration increased for both cpTi and TiZr (p?<?0.05). The maximum effect was observed with the 0.5% concentration. Confocal microscopy images suggested an increase in the number of dead bacterial cells with increased chlorhexidine concentration. The biofilm pH increased after chlorhexidine exposure (p?<?0.05). Chlorhexidine showed an antimicrobial dose-response effect in controlling biofilm on cpTi and TiZr. 0.5% chlorhexidine can be used to achieve the maximum antimicrobial effect on both materials.     

The Population Decline and Extinction of Darwin’s Frogs

Darwin’s frogs (Rhinoderma darwinii and R. rufum) are two species of mouth-brooding frogs from Chile and Argentina. Here, we present evidence on the extent of declines, current distribution and conservation status of Rhinoderma spp.; including information on abundance, habitat and threats to extant Darwin’s frog populations. All known archived Rhinoderma specimens were examined in museums in North America, Europe and South America. Extensive surveys were carried out throughout the historical ranges of R. rufum and R. darwinii from 2008 to 2012. Literature review and location data of 2,244 archived specimens were used to develop historical distribution maps for Rhinoderma spp. Based on records of sightings, optimal linear estimation was used to estimate whether R. rufum can be considered extinct. No extant R. rufum was found and our modelling inferred that this species became extinct in 1982 (95% CI, 1980–2000). Rhinoderma darwinii was found in 36 sites. All populations were within native forest and abundance was highest in Chiloé Island, when compared with Coast, Andes and South populations. Estimated population size and density (five populations) averaged 33.2 frogs/population (range, 10.2–56.3) and 14.9 frogs/100 m² (range, 5.3–74.1), respectively. Our results provide further evidence that R. rufum is extinct and indicate that R. darwinii has declined to a much greater degree than previously recognised. Although this species can still be found across a large part of its historical range, remaining populations are small and severely fragmented. Conservation efforts for R. darwinii should be stepped up and the species re-classified as Endangered.     

Finding protein-protein interaction patterns by contact map matching

We propose a novel method for defining patterns of contacts present in protein-protein complexes. A new use of the traditional contact maps (more frequently used for representation of the intra-chain contacts) is presented for analysis of inter-chain contacts. Using an algorithm based on image processing techniques, we can compare protein-protein interaction maps and also obtain a dissimilarity score between them. The same algorithm used to compare the maps can align the contacts of all the complexes and be helpful in the determination of a pattern of conserved interactions at the interfaces. We present an example for the application of this method by analyzing the pattern of interaction of bovine pancreatic trypsin inhibitors and trypsins, chymotrypsins, a thrombin, a matriptase, and a kallikrein - all classified as serine proteases. We found 20 contacts conserved in trypsins and chymotrypsins and 3 specific ones are present in all the serine protease complexes studied. The method was able to identify important contacts for the protein family studied and the results are in agreement with the literature.     

Biology of Bactrocera carambolae (Diptera: Tephritidae) on four hosts

Bactrocera carambolae is a quarantine pest found in Brazil, restricted to the states of Amapá, Pará and Roraima. This fruit fly can potentially cause extensive socioeconomic and environmental damage in the country, if it disperse into areas where fruit is grown for exporting. The objective of this work was to study the biology of B. carambolae on fruits of Averrhoa carambola L. (Oxalidaceae), Psidium guajava L. (Myrtaceae), Spondias mombin L. (Anacardiaceae) and Eugenia stipitata McVaugh (Myrtaceae). The following parameters were investigated: duration of egg-larva, pupal, egg-adult, pre-oviposition, oviposition and post-oviposition periods, pupal weight and viability, sex ratio, fecundity, fertility and longevity. All parameters except pupal weight, oviposition and post-oviposition period, egg fertility and sex ratio were influenced by the host plant on which the larvae were reared. The carambola fruit fly completes its development on all those hosts studied here, with the highest fecundities on A. carambola and P. guajava.     

Creation of genome-wide protein expression libraries using random activation of gene expression

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.