Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.     

Sex differences in drug conjugation and their consequences for drug toxicity. Sulfation, glucuronidation and glutathione conjugation

Twenty-six flavonoids and related compounds were screened for their ability to modulate microsome mediated covalent adduct formation between [3H]benzo[a]pyrene ([3H]BP) and DNA in vitro. Some of these flavonoids, notably robinetin, quercetin, isorhamnetin and kaempferol were observed to inhibit the adduct formation significantly at very low levels. The unsubstituted flavone and some of the other flavonoids moderately inhibited this adduct formation, while some flavonoids were inactive, viz., most of the isoflavonoids and methylether derivatives of polyhydroxylated flavonoids. Structural features contributory towards the inhibitory activity of flavonoids appeared to be hydroxyl groups in 3 position of C ring, 5,7-positions of A ring and 3',4'- and 5'-positions of B ring. Methylation or glycosylation of hydroxyl group rendered the flavonoid less active or inactive. Flavanones, with saturated 2,3 double bond, were also inactive. Metabolic activation of BP to proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BP-7,8-dihydrodiol) was also measured in presence of some of these flavonoids. The extent of inhibition of metabolism by these flavonoids did not correlate with their ability to inhibit the adduct formation. Thus, suppression of metabolism did not appear to be a major contributory factor towards inhibition of adduct formation. The solvolysis in aqueous dioxane of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE I), the ultimate carcinogen of BP, was accelerated in presence of selected flavonoids. Inactivation of BPDE I, therefore, appeared to be the major mechanism by which some of these flavonoids inhibited the adduct formation between BP and DNA, and this could be the basis for the anti-carcinogenic nature of these flavonoids.     

Predicting and Analyzing Interactions between Mycobacterium tuberculosis and Its Human Host

The outcome of infection by Mycobacterium tuberculosis (Mtb) depends greatly on how the host responds to the bacteria and how the bacteria manipulates the host, which is facilitated by protein–protein interactions. Thus, to understand this process, there is a need for elucidating protein interactions between human and Mtb, which may enable us to characterize specific molecular mechanisms allowing the bacteria to persist and survive under different environmental conditions. In this work, we used the interologs method based on experimentally verified intra-species and inter-species interactions to predict human-Mtb functional interactions. These interactions were further filtered using known human-Mtb interactions and genes that are differentially expressed during infection, producing 190 interactions. Further analysis of the subcellular location of proteins involved in these human-Mtb interactions confirms feasibility of these interactions. We also conducted functional analysis of human and Mtb proteins involved in these interactions, checking whether these proteins play a role in infection and/or disease, and enriching Mtb proteins in a previously predicted list of drug targets. We found that the biological processes of the human interacting proteins suggested their involvement in apoptosis and production of nitric oxide, whereas those of the Mtb interacting proteins were relevant to the intracellular environment of Mtb in the host. Mapping these proteins onto KEGG pathways highlighted proteins belonging to the tuberculosis pathway and also suggested that Mtb proteins might use the host to acquire nutrients, which is in agreement with the intracellular lifestyle of Mtb. This indicates that these interactions can shed light on the interplay between Mtb and its human host and thus, contribute to the process of designing novel drugs with new biological mechanisms of action.     

Dengue Virus Co-opts UBR4 to Degrade STAT2 and Antagonize Type I Interferon Signaling

An estimated 50 million dengue virus (DENV) infections occur annually and more than forty percent of the human population is currently at risk of developing dengue fever (DF) or dengue hemorrhagic fever (DHF). Despite the prevalence and potential severity of DF and DHF, there are no approved vaccines or antiviral therapeutics available. An improved understanding of DENV immune evasion is pivotal for the rational development of anti-DENV therapeutics. Antagonism of type I interferon (IFN-I) signaling is a crucial mechanism of DENV immune evasion. DENV NS5 protein inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation. Only proteolytically-processed NS5 can efficiently mediate STAT2 degradation, though both unprocessed and processed NS5 bind STAT2. Here we identify UBR4, a 600-kDa member of the N-recognin family, as an interacting partner of DENV NS5 that preferentially binds to processed NS5. Our results also demonstrate that DENV NS5 bridges STAT2 and UBR4. Furthermore, we show that UBR4 promotes DENV-mediated STAT2 degradation, and most importantly, that UBR4 is necessary for efficient viral replication in IFN-I competent cells. Our data underscore the importance of NS5-mediated STAT2 degradation in DENV replication and identify UBR4 as a host protein that is specifically exploited by DENV to inhibit IFN-I signaling via STAT2 degradation.     

The importance of denitrification for N2O emissions from an N-saturated forest in SW China: results from in situ 15N labeling experiments

Long-term elevated atmogenic deposition (~5 g m^?2 year^?1) of reactive nitrogen (N) causes N saturation in forests of subtropical China which may lead to high nitrous oxide (N₂O) emissions. Recently, we found high N₂O emission rates (up to 1,730 μg N₂O–N m^?2 h^?1) during summer on well-drained acidic acrisols (pH = 4.0) along a hill slope in the forested Tieshanping catchment, Chongqing, southwest China. Here, we present results from an in situ ¹⁵N–NO₃ ^? labeling experiment to assess the contribution of nitrification and denitrification to N₂O emissions in these soils. Two loads of 99 at.% K¹⁵NO₃ (equivalent to 0.2 and 1.0 g N m^?2) were applied as a single dose to replicated plots at two positions along the hill slope (at top and bottom, respectively) during monsoonal summer. During a 6-day period after label application, we found that 71–100 % of the emitted N₂O was derived from the labeled NO₃ ^? pool irrespective of slope position. Based on this, we assume that denitrification is the dominant process of N₂O formation in these forest soils. Within 6 days after label addition, the fraction of the added ¹⁵N–NO₃ ^? emitted as ¹⁵N–N₂O was highest at the low-N addition plots (0.2 g N m^?2), amounting to 1.3 % at the top position of the hill slope and to 3.2 % at the bottom position, respectively. Our data illustrate the large potential of acid forest soils in subtropical China to form N₂O from excess NO₃ ^? most likely through denitrification.     

Predictive Value of the Tuberculin Skin Test among Newly Arriving Immigrants

Rationale

Screening and treating newly arriving immigrants for latent tuberculosis infection (LTBI) in low-incidence countries could be promising to reduce the tuberculosis incidence among this population. The effectiveness of screening with the tuberculin skin test (TST) is unknown.

Objectives

To estimate the risk of progression to tuberculosis within two years after entry, stratified by TST result at entry.

Methods

In a case-base design, we determined the prevalence of TST positives (10 mm and 15 mm) among a representative cohort of immunocompetent immigrants (n = 643) aged ≥18 years who arrived between April 2009 and March 2011 in the Netherlands (base cohort). Immigrants who progressed to tuberculosis within two years after arrival in 2005, 2006 or 2007 were extracted from the Netherlands Tuberculosis Register (case source cohort). The prevalence of TST positives from the base cohort was projected on the case source cohort to estimate the risk of progression to active tuberculosis by using Bayesian analyses to adjust for the sensitivity of the TST and Poisson regression analyses to take into account the random error of the number of extracted cases.

Results

The prevalence of TST positives was 42% and 23% for a cut-off value of 10 mm and 15 mm, respectively. The overall risk of progression to tuberculosis if TST positive was 238 per 100,000 population (95% CI 151–343) and 295 per 100,000 population (95% CI 161–473) for a cut-off value of ≥10 mm and ≥15 mm, respectively. The corresponding risk for TST negatives was 19 (95% CI 0–59) and 58 (95% CI 25–103).

Conclusion

The TST has the discriminatory ability to differentiate between individuals at low and high risk of disease.