Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples

Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases. It is important to assess the oxidative injury in vivo accurately and inclusively. We have developed an improved method for the measurement of in vivo lipid peroxidation by using a single plasma or liver sample, where total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)), total hydroxyoctadecadienoic acids (tHODEs), total hydroxyeicosatetraenoic acids (tHETEs), and total 7-hydroxycholesterol (t7-OHCh), as well as their parent molecules linoleic acid (t18:2) and cholesterol (tCh), are determined by LC-MS/MS (for t8-iso-PGF(2alpha), tHODE, and tHETE) and GC-MS (for t7-OHCh, t18:2, and tCh) analyses. The plasma and liver samples from human are reduced with sodium borohydride and saponified by potassium hydroxide after the addition of heavy isotopic standards. After extraction by chloroform/ethyl acetate (CHCl(3)/CH(3)COOC(2)H(5), 4:1), they are analyzed without any further sample processing. We applied this method to hepatitis C virus-infected patients (n=8, plasma and liver), hepatitis B virus-infected patients (n=2, plasma and liver), and controls (virus free, n=8, plasma and liver). It was found that in the plasma of patients and controls, the concentrations of oxidized lipids decreased in the following order: tHODE tHETE t7-OHCh > t8-iso-PGF(2alpha). As expected, the virus clearly increased these concentrations. The ratio of stereoisomers of HODE [(E,E)-HODE/(E,Z)-HODE], which reflects the antioxidant capacity in vivo, can also be determined by this method. A significant decrease in the stereoisomer ratio for the liver of patients was observed, indicating liver dysfunction. t8-iso-PGF(2alpha), tHODE, tHETE, and t7-OHCh are measured satisfactorily and inclusively by the current method from biological fluids and tissues, and they can account for a large portion of oxidized lipids in vivo.     

Actigraphic estimates of sleep and the sleep-wake rhythm,and 6-sulfatoxymelatonin levels in healthy Dutch children

ABSTRACT

Sleep and the sleep-wake rhythm are essential for children’s health and well-being, yet reference values are lacking. This study therefore aimed to assess actigraphic estimates of sleep and the 24-h sleep-wake rhythm, as well as 6-sulfatoxymelatonin (aMT6s) levels in healthy children of different age groups. Additionally, relationships between the outcomes and sex, highest parental educational level (as an indication of socioeconomic status (SES)), and body-mass-index (BMI) were explored. In this cross-sectional study, healthy Dutch children (2–18 years) wore an actigraph (GT3x) for 7 consecutive days, collected first-morning void urine and completed a sleep log and sociodemographic questionnaire. Actigraphically estimated sleep variables were sleep onset latency (SOL), sleep efficiency (SE), total sleep time (TST), and wake after sleep onset (WASO). Non-parametric sleep-wake rhythm variables were intradaily variability (IV); interdaily stability (IS); the activity counts and timing of the least active 5-h period (L5counts and midpoint) and of the most active 10-h period (M10 counts and midpoint); and the relative amplitude (RA), i.e. the ratio of the difference and the sum of M10 and L5 counts. Finally, creatinine-corrected aMT6s levels were obtained by isotope dilution mass spectrometry. Effects of age group (preschool 2–5 years/school-aged 6–12 years/teenager 13–18 years), sex, highest parental educational level and BMI (Z-scores) were explored. Ninety-four children participated, equally divided across age groups (53% boys). Teenagers slept less, but more efficiently, than younger children, while their 24 h sleep-wake rhythm was the least stable and most fragmented (likely due to fragmentation of daytime activity). Additionally, aMT6s levels significantly declined over the age groups. Children from highly educated parents had lower sleep efficiency, but a more stable sleep-wake rhythm. Finally, sex or increase in BMI was not associated with any of the outcomes in this study. In conclusion, this study provides reference values of healthy children across different age groups and different sociodemographic factors. In the future, this information may help to better interpret outcomes in clinical populations.     

Purification and Characterization of Kynurenine Aminotransferase I from Human Brain

Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l -kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute K _m of 2.0 m M and 10.0 m M for l -kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l -glutamine, l -phenylalanine, and l -tryptophan, using either pyruvate (1 m M ) or 2-oxoisocaproate (1 m M ) as a cosubstrate. l -Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate ( K _i = 480 µ M ) and competitively with regard to l -kynurenine ( K _i = 200 µ M ). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.     

Acclimatization and response of minipigs toward humans.

As a first step toward making an efficient acclimatization methodology for minipigs, the reaction of G?ttingen minipigs, 3-24 months of age, toward humans was investigated. All minipigs were kept in an individual cage, and the reaction toward humans (acclimatization index) was evaluated by simple observations. The acclimatization index was evaluated as the total number of points scored (0-30 points) based on the following criteria: (1) the position of the minipig when the cage door was opened; (2) the reaction of the minipig when the observer approached; and (31 the reaction of the minipig when the observer touched it. Subsequently, each animal was ranked by total points scored: 30 points = AA, 20 points < or = A < 30 points, 10 points < or = B < 20 points, 0 points < or = C < 10 points. Based on this evaluation, the reactions of minipigs under three conditions were investigated. The following findings were confirmed: first, minipig reaction to humans was influenced by monthly age; second, taming was possible under ordinary conditions of care, but we had to wait until 10 months of age on average for this to occur; third, if simple contact was made during care time, minipigs became tame within less than 4 weeks after the commencement of contact. We therefore consider it possible to artificially control the reaction of minipigs toward humans, and to make minipigs more available for experiments by adding control of the hereditary factors that influence this reaction.     

Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod‐shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re‐assemble, and MreB‐free zones were subsequently observed in the cytoplasmic membrane. These MreB‐free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y‐shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction.     

Arg16/Gly beta2-adrenergic receptor polymorphism alters the cardiac output response to isometric exercise.

Normotensive adults homozygous for glycine (Gly) of the Arg16/Gly beta2-adrenergic-receptor polymorphism have 1) greater forearm beta2-receptor mediated vasodilation and 2) a higher heart rate (HR) response to isometric handgrip than arginine (Arg) homozygotes. To test the hypothesis that the higher HR response in Gly16 subjects serves to maintain the pressor response [increased cardiac output (CO)] in the setting of augmented peripheral vasodilation to endogenous catecholamines, we measured continuous HR (ECG), arterial pressure (Finapres), and CO (transthoracic echocardiography) during isometric, 40% submaximal handgrip to fatigue in healthy subjects homozygous for Gly (n = 30; mean age +/- SE: 30 +/- 1.2, 13 women) and Arg (n = 17, age 30 +/- 1.6, 11 women). Resting data were similar between groups. Handgrip produced similar increases in arterial pressure and venous norepinephrine and epinephrine concentrations; however, HR increased more in the Gly group (60.1 +/- 4.3% increase from baseline vs. 45.5 +/- 3.9%, P = 0.03), and this caused CO to be higher (Gly: 7.6 +/- 0.3 l/m vs. Arg: 6.5 +/- 0.3 l/m, P = 0.03), whereas the decrease in systemic vascular resistance in the Gly group did not reach significance (P = 0.09). We conclude that Gly16 homozygotes generate a higher CO to maintain the pressor response to handgrip. The influence of polymorphic variants in the beta2-adrenergic receptor gene on the cardiovascular response to sympathoexcitation may have important implications in the development of hypertension and heart failure.     

Partial proteasome inhibition in human fibroblasts triggers accelerated M1 senescence or M2 crisis depending on p53 and Rb status

Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression are reduced during aging and replicative senescence. Its activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs), whereas partial proteasome inhibition triggers an irreversible premature senescent state in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence (RS and PS, respectively), in this study we investigated their implication in proteasome inhibition-mediated PS. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we reveal that proteasome activity inhibition to levels normally found in senescent human cells results in immediate growth arrest and/or moderate increase of apoptotic death. These effects are independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated PS can only be initiated and maintained in the presence of functional p53. More specifically, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the continuous interplay between the integrity of proteasome function, senescence and cell survival.