Protein markers of ischemic insult in brain endothelial cells identified using 2D gel electrophoresis and ICAT-based quantitative proteomics

The blood-brain barrier (BBB) is formed by endothelial cells of cerebral microvessels sealed by tight junctions. Ischemic brain injury is known to initiate a series of biochemical and molecular processes that lead to the disruption of the BBB, development of vascular inflammation, and subsequent neurovascular remodeling including angiogenesis. Molecular effectors of these changes are multiple and are regulated in a dynamic fashion. The current study was designed to analyze changes in cellular and secreted proteins in rat brain endothelial cells (BEC) exposed to ischemic insult in vitro using two complementary quantitative proteomic approaches: two-dimensional gel electrophoresis (2DE) and isotope-coded affinity tag (ICAT)-based proteomics. We show a comprehensive qualitative and quantitative comparison between the two proteomic methods applied to the same experimental system with respect to their reproducibility, specificity, and the type of proteins identified. In total, >160 proteins showed differential expression in response to the ischemic insult, with 38 identified by 2DE and 138 by ICAT. Only 15 proteins were commonly identified. ICAT showed superior reproducibility over 2DE and was more suitable for detecting small, large, basic, hydrophobic, and secreted proteins than 2DE. However, positive identification of proteins by MS/MS was more reliably done using a 2DE-based method compared to ICAT. Changes in proteins involved in nucleic acid, protein, and carbohydrate metabolism, signal transduction, cell structure, adhesion and motility, immunity and defense, cell cycle, and apoptosis were observed. The functional significance of observed protein changes was evaluated through a multifaceted protein classification and validation process, which included literature mining and comparative evaluation of protein changes in analogous in vitro and in vivo ischemia models. The comparative analyses of protein changes between the in vitro and in vivo models demonstrated a significant correlative relationship, emphasizing the 'translational' value of in vitro endothelial models in neurovascular research.     

Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3

Enzymatic incorporation of a halogen atom is a common feature in the biosyntheses of more than 4,500 natural products. Halogenation of unactivated carbon centers in the biosyntheses of several compounds of nonribosomal peptide origin is carried out by a class of mononuclear nonheme iron enzymes that require alpha-ketoglutarate (alphaKG, 1), chloride and oxygen. To investigate the ability of these enzymes to functionalize unactivated methyl groups, we characterized the chlorination of the gamma-methyl substituent of L-2-aminobutyric acid (L-Aba, 2) attached to the carrier protein CytC2 by iron halogenase (CytC3) from soil Streptomyces sp. We identified an intermediate state comprising two high-spin Fe(IV) complexes in rapid equilibrium. At least one of the Fe(IV) complexes abstracts hydrogen from the substrate. The demonstration that chlorination proceeds through an Fe(IV) intermediate that cleaves a C-H bond reveals the mechanistic similarity of aliphatic halogenases to the iron- and alphaKG-dependent hydroxylases.     

The ecophysiology of seed persistence: a mechanistic view of the journey to germination or demise

Seed persistence is the survival of seeds in the environment once they have reached maturity. Seed persistence allows a species, population or genotype to survive long after the death of parent plants, thus distributing genetic diversity through time. The ability to predict seed persistence accurately is critical to inform long‐term weed management and flora rehabilitation programs, as well as to allow a greater understanding of plant community dynamics. Indeed, each of the 420000 seed‐bearing plant species has a unique set of seed characteristics that determine its propensity to develop a persistent soil seed bank. The duration of seed persistence varies among species and populations, and depends on the physical and physiological characteristics of seeds and how they are affected by the biotic and abiotic environment. An integrated understanding of the ecophysiological mechanisms of seed persistence is essential if we are to improve our ability to predict how long seeds can survive in soils, both now and under future climatic conditions. In this review we present an holistic overview of the seed, species, climate, soil, and other site factors that contribute mechanistically to seed persistence, incorporating physiological, biochemical and ecological perspectives. We focus on current knowledge of the seed and species traits that influence seed longevity under ex situ controlled storage conditions, and explore how this inherent longevity is moderated by changeable biotic and abiotic conditions in situ, both before and after seeds are dispersed. We argue that the persistence of a given seed population in any environment depends on its resistance to exiting the seed bank via germination or death, and on its exposure to environmental conditions that are conducive to those fates. By synthesising knowledge of how the environment affects seeds to determine when and how they leave the soil seed bank into a resistance–exposure model, we provide a new framework for developing experimental and modelling approaches to predict how long seeds will persist in a range of environments.     

Reversible acetylation of metabolic enzymes celebration: SIRT2 and p300 join the party

Compelling evidence suggests that metabolic pathways are coordinated through reversible acetylation of metabolic enzymes in response to nutrient availability. In this issue of Molecular Cell, Jiang et al. (2011) show that the rate-limiting enzyme in gluconeogenesis, phosphoenolpyruvate carboxykinase 1, is regulated through reversible acetylation by SIRT2 and p300.     

Evidence for time-of-day dependent effect of neurotoxic dorsomedial hypothalamic lesions on food anticipatory circadian rhythms in rats

The dorsomedial hypothalamus (DMH) is a site of circadian clock gene and immediate early gene expression inducible by daytime restricted feeding schedules that entrain food anticipatory circadian rhythms in rats and mice. The role of the DMH in the expression of anticipatory rhythms has been evaluated using different lesion methods. Partial lesions created with the neurotoxin ibotenic acid (IBO) have been reported to attenuate food anticipatory rhythms, while complete lesions made with radiofrequency current leave anticipatory rhythms largely intact. We tested a hypothesis that the DMH and fibers of passage spared by IBO lesions play a time-of-day dependent role in the expression of food anticipatory rhythms. Rats received intra-DMH microinjections of IBO and activity and body temperature (T(b)) rhythms were recorded by telemetry during ad-lib food access, total food deprivation and scheduled feeding, with food provided for 4-h/day for 20 days in the middle of the light period and then for 20 days late in the dark period. During ad-lib food access, rats with DMH lesions exhibited a lower amplitude and mean level of light-dark entrained activity and T(b) rhythms. During the daytime feeding schedule, all rats exhibited food anticipatory activity and T(b) rhythms that persisted during 2 days without food in constant dark. In some rats with partial or total DMH ablation, the magnitude of the anticipatory rhythm was weak relative to most intact rats. When mealtime was shifted to the late night, the magnitude of the food anticipatory activity rhythms in these cases was restored to levels characteristic of intact rats. These results confirm that rats can anticipate scheduled daytime or nighttime meals without the DMH. Improved anticipation at night suggests a modulatory role for the DMH in the expression of food anticipatory activity rhythms during the daily light period, when nocturnal rodents normally sleep.     

Development and validation of a risk model for prediction of hazardous alcohol consumption in general practice attendees: the predictAL study

Background

Little is known about the risk of progression to hazardous alcohol use in people currently drinking at safe limits. We aimed to develop a prediction model (predictAL) for the development of hazardous drinking in safe drinkers.

Methods

A prospective cohort study of adult general practice attendees in six European countries and Chile followed up over 6 months. We recruited 10,045 attendees between April 2003 to February 2005. 6193 European and 2462 Chilean attendees recorded AUDIT scores below 8 in men and 5 in women at recruitment and were used in modelling risk. 38 risk factors were measured to construct a risk model for the development of hazardous drinking using stepwise logistic regression. The model was corrected for over fitting and tested in an external population. The main outcome was hazardous drinking defined by an AUDIT score ≥8 in men and ≥5 in women.

Results

69.0% of attendees were recruited, of whom 89.5% participated again after six months. The risk factors in the final predictAL model were sex, age, country, baseline AUDIT score, panic syndrome and lifetime alcohol problem. The predictAL model''s average c-index across all six European countries was 0.839 (95% CI 0.805, 0.873). The Hedge''s g effect size for the difference in log odds of predicted probability between safe drinkers in Europe who subsequently developed hazardous alcohol use and those who did not was 1.38 (95% CI 1.25, 1.51). External validation of the algorithm in Chilean safe drinkers resulted in a c-index of 0.781 (95% CI 0.717, 0.846) and Hedge''s g of 0.68 (95% CI 0.57, 0.78).

Conclusions

The predictAL risk model for development of hazardous consumption in safe drinkers compares favourably with risk algorithms for disorders in other medical settings and can be a useful first step in prevention of alcohol misuse.     

Cytotoxic activity of acridin-3,6-diyl dithiourea hydrochlorides in human leukemia line HL-60 and resistant subline HL-60/ADR

A series of acridin-3,6-diyl dithiourea hydrochloride derivatives (alkyl-AcrDTU) was prepared and tested against sensitive and drug resistant leukemia cell lines for their cytotoxic/cytostatic activity. The products (ethyl-, n-propyl-, n-butyl-, n-pentyl-AcrDTU) showed high DNA binding affinity via intercalation (K = 7.6 ? 2.9 × 10⁵ M^?1). All derivatives inhibited proliferation of HL-60 cells and its resistant subline HL-60/ADR, unexpectedly the resistant subline was more sensitive than the parental one (IC₅₀ = 3.5 μM, 48-treatment of HL-60/ADR with pentyl-AcrDTU). Cytotoxicity of tested compounds was associated with their DNA-binding properties and the level of intracellular thiols has been changed in the presence of AcrDTU.     

Circadian clocks for all meal-times: anticipation of 2 daily meals in rats

Anticipation of a daily meal in rats has been conceptualized as a rest-activity rhythm driven by a food-entrained circadian oscillator separate from the pacemaker generating light-dark (LD) entrained rhythms. Rats can also anticipate two daily mealtimes, but whether this involves independently entrained oscillators, one 'continuously consulted' clock, cue-dependent non-circadian interval timing or a combination of processes, is unclear. Rats received two daily meals, beginning 3-h (meal 1) and 13-h (meal 2) after lights-on (LD 14:10). Anticipatory wheel running began 68±8 min prior to meal 1 and 101±9 min prior to meal 2 but neither the duration nor the variability of anticipation bout lengths exhibited the scalar property, a hallmark of interval timing. Meal omission tests in LD and constant dark (DD) did not alter the timing of either bout of anticipation, and anticipation of meal 2 was not altered by a 3-h advance of meal 1. Food anticipatory running in this 2-meal protocol thus does not exhibit properties of interval timing despite the availability of external time cues in LD. Across all days, the two bouts of anticipation were uncorrelated, a result more consistent with two independently entrained oscillators than a single consulted clock. Similar results were obtained for meals scheduled 3-h and 10-h after lights-on, and for a food-bin measure of anticipation. Most rats that showed weak or no anticipation to one or both meals exhibited elevated activity at mealtime during 1 or 2 day food deprivation tests in DD, suggesting covert operation of circadian timing in the absence of anticipatory behavior. A control experiment confirmed that daytime feeding did not shift LD-entrained rhythms, ruling out displaced nocturnal activity as an explanation for daytime activity. The results favor a multiple oscillator basis for 2-meal anticipatory rhythms and provide no evidence for involvement of cue-dependent interval timing.