Regulated cellular partitioning of SR protein-specific kinases in mammalian cells

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.     

Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats

Energy restriction (ER), without malnutrition, is the only environmental intervention that consistently increases maximum life span in laboratory rodents. One theory proposes that a reduction in energy expenditure and reactive oxygen species production is the mechanism responsible for this action of ER. To further test this theory, proton leak, H2O2 production, lipid peroxidation, and protein carbonyls were measured in mitochondria from FBNF1 rats fed either a control or 40% ER diet (onset at 6 mo of age). Liver mitochondria were isolated at 7 and 12 mo of age. Liver weight decreased 25 and 36% at 1 and 6 mo of ER, respectively (P < 0.05). ER resulted in an increase (P < 0.05) in percent total polyunsaturates, n-6 polyunsaturates, and total unsaturates (6 mo only) in mitochondrial lipids. These changes, however, were not associated with significant alterations in mitochondrial function. State 4 respiration and membrane potential were not different (P > 0.05) between groups at either assessment period. Similarly, proton leak kinetics were not different between control and ER animals. Top-down metabolic control analysis and its extension, elasticity analysis, were used at the 6-mo assessment and revealed no difference in control of the oxidative phosphorylation system between control and ER rats. H2O2 production with either succinate or pyruvate/malate substrates was also not different (P > 0.05) between groups at either time point. In conclusion, ER did not alter proton leak or H2O2 production at this age or stage of restriction in liver.     

Long-term caloric restriction increases UCP3 content but decreases proton leak and reactive oxygen species production in rat skeletal muscle mitochondria

Calorie restriction (CR) without malnutrition increases life span and delays the onset of a variety of diseases in a wide range of animal species. However, the mechanisms responsible for the retardation of aging with CR are poorly understood. We proposed that CR may act, in part, by inducing a hypometabolic state characterized by decreased reactive oxygen species (ROS) production and mitochondrial proton leak. Here, we examine the effects of long-term CR on whole animal energetics as well as muscle mitochondrial energetics, ROS production, and ROS damage. CR was initiated in male FBNF1 rats at 6 mo of age and continued for 12 or 18 mo. Mean whole body VO2 was 34.6 (P < 0.01) and 35.6% (P < 0.001) lower in CR rats than in controls after 12 and 18 mo of CR, respectively. Body mass-adjusted VO2 was 11.1 and 29.5% lower (both P < 0.05) in CR rats than in controls after 12 and 18 mo of CR. Muscle mitochondrial leak-dependent (State 4) respiration was decreased after 12 mo compared with controls; however, after 18 mo of CR, there were slight but not statistically significant differences. Proton leak kinetics were affected by 12 mo of CR such that leak-dependent respiration was lower in CR mitochondria only at protonmotive force values exceeding 170 mV. Mitochondrial H2O2 production and oxidative damage were decreased by CR at both time points and increased with age. Muscle UCP3 protein content increased with long-term CR, consistent with a role in protection from ROS but inconsistent with the observed decrease or no change in proton leak.     

Kinetic basis for activation of CDK2/cyclin A by phosphorylation

The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr(160) in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr(160) does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group.     

Injuries,Death, and Disability Associated with 11 Years of Conflict in Baghdad,Iraq: A Randomized Household Cluster Survey

Background

The objective of this study was to characterize injuries, deaths, and disabilities arising during 11 years of conflict in Baghdad.

Methods

Using satellite imagery and administrative population estimated size for Baghdad, 30 clusters were selected, proportionate to population size estimates. Interviews were conducted during April and May 2014 in 900 households containing 5148 persons. Details about injuries and disabilities occurring from 2003 through May 2014 and resultant disabilities were recorded.

Findings

There were 553 injuries reported by Baghdad residents, 225 of which were intentional, and 328 unintentional. For intentional injuries, the fatality rate was 39.1% and the disability rate 56.0%. Gunshots where the major cause of injury through 2006 when blasts/explosions became the most common cause and remained so through 2014. Among unintentional injuries, the fatality rate was 7.3% and the disability rate 77.1%. The major cause of unintentional injuries was falls (131) which have increased dramatically since 2008, followed by traffic related injuries (81), which have steadily increased. The proportion of injuries ending in disabilities remained fairly constant through the survey period.

Interpretation

Intentional injuries added substantially to the burden of unintentional injuries for the population. For Baghdad, the phases of the Iraqi conflict are reflected in the patterns of injuries and consequent deaths reported. The scale of injuries during conflict is most certainly under-reported. Difficulties recalling injuries in a survey covering 11 years is a limitation, but it is likely that minor injuries were under-reported more than severe injuries. The in- and out-migration of Baghdad populations likely had effects on the events reported which we could not measure or estimate. Damage to the health infrastructure and the flight of health workers may have contributed to mortality and morbidity. Civilian injuries as well as mortality should be measured during conflicts, though not currently done.     

Mortality in Iraq Associated with the 2003–2011 War and Occupation: Findings from a National Cluster Sample Survey by the University Collaborative Iraq Mortality Study

Background

Previous estimates of mortality in Iraq attributable to the 2003 invasion have been heterogeneous and controversial, and none were produced after 2006. The purpose of this research was to estimate direct and indirect deaths attributable to the war in Iraq between 2003 and 2011.

Methods and Findings

We conducted a survey of 2,000 randomly selected households throughout Iraq, using a two-stage cluster sampling method to ensure the sample of households was nationally representative. We asked every household head about births and deaths since 2001, and all household adults about mortality among their siblings. We used secondary data sources to correct for out-migration. From March 1, 2003, to June 30, 2011, the crude death rate in Iraq was 4.55 per 1,000 person-years (95% uncertainty interval 3.74–5.27), more than 0.5 times higher than the death rate during the 26-mo period preceding the war, resulting in approximately 405,000 (95% uncertainty interval 48,000–751,000) excess deaths attributable to the conflict. Among adults, the risk of death rose 0.7 times higher for women and 2.9 times higher for men between the pre-war period (January 1, 2001, to February 28, 2003) and the peak of the war (2005–2006). We estimate that more than 60% of excess deaths were directly attributable to violence, with the rest associated with the collapse of infrastructure and other indirect, but war-related, causes. We used secondary sources to estimate rates of death among emigrants. Those estimates suggest we missed at least 55,000 deaths that would have been reported by households had the households remained behind in Iraq, but which instead had migrated away. Only 24 households refused to participate in the study. An additional five households were not interviewed because of hostile or threatening behavior, for a 98.55% response rate. The reliance on outdated census data and the long recall period required of participants are limitations of our study.

Conclusions

Beyond expected rates, most mortality increases in Iraq can be attributed to direct violence, but about a third are attributable to indirect causes (such as from failures of health, sanitation, transportation, communication, and other systems). Approximately a half million deaths in Iraq could be attributable to the war. Please see later in the article for the Editors'' Summary     

Limited RNA Editing in Exons of Mouse Liver and Adipose

Several studies have investigated RNA–DNA differences (RDD), presumably due to RNA editing, with conflicting results. We report a rigorous analysis of RDD in exonic regions in mice, taking into account critical biases in RNA-Seq analysis. Using deep-sequenced F1 reciprocal inbred mice, we mapped 40 million RNA-Seq reads per liver sample and 180 million reads per adipose sample. We found 7300 apparent hepatic RDDs using a multiple-site mapping procedure, compared with 293 RDD found using a unique-site mapping procedure. After filtering for repeat sequence, splice junction proximity, undirectional strand, and extremity read bias, 63 RDD remained. In adipose tissue unique-site mapping identified 1667 RDD, and after applying the same four filters, 188 RDDs remained. In both tissues, the filtering procedure increased the proportion of canonical (A-to-I and C-to-U) editing events. The genomic DNA of 12 RDD sites among the potential 63 hepatic RDD was tested by Sanger sequencing, three of which proved to be due to unreferenced SNPs. We validated seven liver RDD with Sequenom technology, including two noncanonical, Gm5424 C-to-I(G) and Pisd I(G)-to-A RDD. Differences in diet, sex, or genetic background had very modest effects on RDD occurrence. Only a small number of apparent RDD sites overlapped between liver and adipose, indicating a high degree of tissue specificity. Our findings underscore the importance of properly filtering for bias in RNA-Seq investigations, including the necessity of confirming the DNA sequence to eliminate unreferenced SNPs. Based on our results, we conclude that RNA editing is likely limited to hundreds of events in exonic RNA in liver and adipose.     

Caloric restriction influences hydrogen peroxide generation in mitochondrial sub-populations from mouse liver

Calorie restriction (CR) has been shown to decrease H₂O₂ production in liver mitochondria, although it is not known if this is due to uniform changes in all mitochondria or changes in particular mitochondrial sub-populations. To address this issue, liver mitochondria from control and CR mice were fractionated using differential centrifugation at 1,000 g, 3,000 g and 10,000 g into distinct populations labeled as M1, M3 and M10, respectively. Mitochondrial protein levels, respiration and H₂O₂ production were measured in each fraction. CR resulted in a decrease in total protein (mg) in each fraction, although this difference disappeared when adjusted for liver weight (mg protein/g liver weight). No differences in respiration (State 3 or 4) were observed between control and CR mice in any of the mitochondrial fractions. CR decreased H₂O₂ production in all fractions when mitochondria respired on succinate (Succ), succ+antimycin A (Succ+AA) or pyruvate/malate+rotenone (P/M+ROT). Thus, CR decreased reactive oxygen species (ROS) production under conditions which stimulate mitochondrial complex I ROS production under both forward (P/M+ROT) and backward (Succ & Succ+AA) electron flow. The results indicate that CR decreases H₂O₂ production in all liver mitochondrial fractions due to a decrease in capacity for ROS production by complex I of the electron transport chain.     

Rat liver xanthine oxidoreductase: effect of adenine on the oxidase and dehydrogenase activities

Xanthine oxidase (XO) and total oxidase plus dehydrogenase (XO+XDH) activities from rat liver were measured in the presence or absence of adenine in extracts prepared with or without DTT/PMSF in homogenization buffer. Presence of adenine in extracts, prepared with or without DTT/PMSF, caused a 45-60% decrease in XO and XO+XDH activities. Removal of adenine by dialysis from extracts prepared with or without DTT/PMSF resulted in the recovery of XO and XO+XDH activities to almost their pre-dialysis control levels. Enzyme activity after 24hr storage at -20 degrees C depended on the presence or absence of DTT/PMSF and adenine, with both XO and XO+XDH activities being lower in extracts with the combined presence of DTT/PMSF and adenine. Incubation of extracts at 37 degrees C for 30 minutes resulted in increased XO and XO+XDH activities, however, adenine-treated samples did not differ from their pre-incubation activities. The molecular mass of the enzyme from control and adenine-treated extracts was unchanged (300 kDa). Adenine-treated extracts prepared with or without DTT/PMSF showed higher D/O ratios in all post-dialysis samples when compared with their pre-dialysis ratios. The results suggest that adenine may play a role in preventing the dehydrogenase to oxidase conversion during extract preparation, storage, overnight dialysis and heat treatment.     

Cryptofaunal diversity in fringing reef rhodoliths

Coral Reefs - Rhodolith distribution, morphology, and cryptofauna have been minimally studied on fringing reefs. We present the first study to examine both rhodolith distribution and associated...