Flotillin‐1 promotes formation of glutamatergic synapses in hippocampal neurons

Synapse malformation underlies numerous neurodevelopmental illnesses, including autism spectrum disorders. Here we identify the lipid raft protein flotillin‐1 as a promoter of glutamatergic synapse formation. We cultured neurons from the hippocampus, a brain region important for learning and memory, and examined them at two weeks in vitro, a time period rich with synapse formation. Double‐label immunocytochemistry of native flot‐1 with glutamatergic and GABAergic synapse markers showed that flot‐1 was preferentially colocalized with the glutamatergic presynaptic marker vesicular glutamate transporter 1 (VGLUT1), compared to the GABAergic presynaptic marker glutamic acid decarboxylase‐65 (GAD‐65). Triple‐label immunocytochemistry of native flot‐1, VGLUT1, and NR1, the obligatory subunit of NMDA receptors, indicates that Flot‐1 was preferentially localized to synaptic rather than extrasynaptic NR1. Furthermore, electrophysiological results using whole‐cell patch clamp showed that Flot‐1 increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs), whereas amplitude and decay kinetics of either type of synaptic current was not affected. Corresponding immunocytochemical data confirmed that the number of glutamatergic synapses increased with flot‐1 overexpression. Overall, our anatomical and physiological results show that flot‐1 enhances the formation of glutamatergic synapses but not GABAergic synapses, suggesting that the role of flot‐1 in neurodevelopmental disorders should be explored. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 875–883, 2010     

Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice

Background

Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents.

Methodology/Principal Findings

We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C^hi/CCR2^hi monocytes. Surprisingly, neutrophils, not Ly6C^lo monocytes, largely replaced Ly6C^hi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.

Conclusion/Significance

These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.     

An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry

We have developed an improved method for the measurement of F2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F2-isoprostane family consists of a series of chemically stable prostaglandin F2 (PGF2)-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Furthermore, 8-epi-PGF2alpha, which is one of the more abundant F2-isoprostanes, is biologically active, being a potent mitogen and vasoconstrictor of rat and rabbit lung and kidney, as well as a partial agonist of platelet aggregation. Measurement of F2-isoprostanes in biological samples is complex and has involved methods which utilize multiple chromatographic steps, including separation by thin-layer chromatography, leading to poor sample recovery. We now present an improved method for the measurement of plasma and urinary F2-isoprostanes using a combination of silica and reverse-phase extraction cartridges, high-performance liquid chromatography (HPLC), and GC-ECNI-MS. Different approaches to the derivatization of the F2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F2-isoprostanes is improved (approx 70% for urine) and the within and between assay reproducibility is 6.7% (n = 23) and 3.7% (n = 3), respectively. The mean urinary excretion of F2-isoprostanes in eight healthy males was 365 +/- 5 pmol/mmol creatinine and in three smokers 981 +/- 138 pmol/mmol creatinine. The mean total (free + esterified) plasma F2-isoprostane concentration was 952 +/- 38 pmol/liter, with a within and between assay reproducibility of 8% (n = 13) and 5.6% (n = 3), respectively. This improved method for the measurement of F2-isoprostanes represents a significant advance in terms of the rapidity and yield in the purification of biological samples. The inclusion of HPLC separation enables improved analysis of F2-isoprostanes by GC-MS. This methodology will assist in defining the role of F2-isoprostanes as in vivo markers of oxidant stress in clinical and experimental settings.     

Macronuclear Molecules Encoding Actins in Spirotrichs

Abstract The nucleotide sequences of 16 newly reported and 8 previously reported actin-encoding macronuclear DNA molecules in spirotrichs have been compared. As described for the eight previously reported molecules, the first 50 bases (noncoding) inside the telomere at both 5′ strands in additional actin molecules are purine-rich. This anomalous base composition might serve as a signal to identify macronuclear molecules in micronuclear DNA during development. The 50-base segment upstream of the ATG in the 5′ leaders of the actin molecules contains extensive, conserved sequence motifs that are possibly promoter elements. The 3′ noncoding trailers contain virtually no conserved sequence motifs. With one exception, the 3′ trailers contain a second stop codon (TGA) 36 bases on average downstream of the primary stop codon. Excluding Moneuplotes crassus, amino acid identities in actin I range from 78 to 100%, with variations distributed nonrandomly along the sequence. Phylogenetic trees based on the actin nucleotide sequences of 22 spirotrichs define the evolutionary relationships of their actin-encoding molecules. The actin phylogeny, while well supported by posterior probabilities, does not always coincide with the phylogeny defined in rDNA analyses or classical taxonomic classifications.     

Effect of eicosapentaenoic acid and docosahexaenoic acid on oxidative stress and inflammatory markers in treated-hypertensive type 2 diabetic subjects

n-3 fatty acids reduce the risk of cardiovascular disease via a number of possible mechanisms. Despite this, there has been concern that these fatty acids may increase lipid peroxidation. The data in vivo are inconclusive, due in part to limitations in the methodologies. In this regard, the measurement of F2-isoprostanes provides a reliable assessment of in vivo lipid peroxidation and oxidant stress. This study aimed to assess the effects of supplementation with purified eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), the two major n-3 fatty acids, on urinary F2-isoprostanes and markers of inflammation, in type 2 diabetic patients. In a double-blind, placebo controlled trial of parallel design, 59 nonsmoking, treated-hypertensive, type 2 diabetic subjects, were randomized to 4 g daily of purified EPA, DHA, or olive oil for 6 weeks, while maintaining their usual diet. F2-isoprostanes, measured using gas chromatography-mass spectrometry in 24 h urines and C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), were measured before and after intervention. Thirty-nine men and 12 women aged 61.2 +/- 1.2 years, with body mass index (BMI), 29.5 +/- 0.5 kg/m2; 24 h blood pressure, 138/73 mmHg; HbA1c, 7.3 +/- 0.1% and fasting glucose, 7.9 +/- 0.2 mmol/l completed the intervention. Baseline urinary F2-isoprostanes were positively associated with HbA1c (p=.011) and fasting glucose (p=.032). Relative to the olive oil group, postintervention urinary F2-isoprostanes were decreased 19% by EPA (p=.017) and 20% by DHA (p=.014). There were no significant changes in CRP, IL-6, and TNF-alpha following EPA or DHA supplementation. In regression analysis, Delta F2-isoprostanes were positively associated with Delta HbA1c (p=.007) independent of treatment group; and with Delta TNF-alpha (p=.034) independent of age, gender, BMI, and treatment group. There were no associations with Delta CRP or Delta IL-6. This study is the first report demonstrating that either EPA or DHA reduce in vivo oxidant stress without changing markers of inflammation, in treated hypertensive, type 2 diabetic subjects.     

Oxidative stress in human hypertension: association with antihypertensive treatment, gender, nutrition, and lifestyle

There is growing evidence that oxidative stress contributes to the pathogenesis of hypertension. Our aim was to measure markers of oxidative stress in hypertensive subjects, and assess the potential confounding influences of antihypertensive therapy, other cardiovascular risk factors, gender, lifestyle, and nutrition. Markers of oxidative stress, including plasma and 24 h urinary F2-isoprostanes, were measured in 70 untreated (men = 43, women = 27) and 85 treated (men = 43, women = 42) hypertensive subjects and 40 normotensive controls (men = 20, women = 20). Overall, F2-isoprostanes were not elevated in hypertensive subjects compared with controls. However, urinary and plasma F2-isoprostanes were significantly lower in treated compared with untreated hypertensive men, but not women. In hypertensive men, the number of antihypertensive drugs taken was inversely associated with both urinary and plasma F2-isoprostanes (p <.05). Self-reported alcohol intake and biomarkers of alcohol consumption (gamma-glutamyl transpeptidase and high-density lipoprotein cholesterol) were positively associated with plasma but not urinary, F2-isoprostanes in men. Several nutrients were independently associated with plasma and urinary F2-isoprostanes in women. The results do not support the hypothesis that treated or untreated hypertensive subjects are under increased oxidative stress compared with normotensive controls.     

Is sexual segregation in the guppy, <Emphasis Type="Italic">Poecilia reticulata</Emphasis>, consistentwith the predation risk hypothesis?

We examined the importance of sex differences in predation risk in generating sexual segregation in the guppy, Poecilia reticulata. We hypothesised that sex differences in predation risk will result in habitat segregation and ultimately social segregation of the sexes, with the more vulnerable sex (males in this case) using safer habitats. In accordance with the predation risk hypothesis we observed sexual segregation in a population associated with high but not low predation risk. Under high predation risk we observed a larger proportion of males in shallow marginal habitats resulting in habitat segregation and ultimately social segregation of the sexes. Furthermore, habitat segregation by sex was associated with habitat segregation by body length with shoals in deeper water having a larger mean body length. Shoaling fish species have been key models in investigating group living, and further research directed towards understanding sexual segregation in other fish species would be valuable.     

Expression of sterol 27-hydroxylase (CYP27A1) enhances cholesterol efflux

Cholesterol efflux from CHOP cells transfected with sterol 27-hydroxylase (CYP27A1) was compared with non-transfected and mock-transfected cells. Transfection caused expression of CYP27A1, formation of 27-hydroxycholesterol, and inhibition of cholesterol biosynthesis. Transfection enhanced cholesterol efflux to apolipoprotein A-I or human plasma by 2-3-fold but did not affect the efflux in the absence of acceptor. The analysis of released sterols revealed that 27-hydroxycholesterol represented only a small proportion of sterols, most of which was non-oxidized cholesterol. Time course and dose dependence studies showed that expression of CYP27A1 in CHOP cells mostly affected the efflux of the "fast" cholesterol pool, and relatively more cholesterol was released with low concentrations of an acceptor. Preincubation of non-transfected cells with exogenous 27-hydroxycholesterol (10(-9) and 10(-7) m) led to the stimulation of cholesterol efflux by 24-60%. Expression of CYP27A1 in CHOP cells did not affect ABCA1 expression and abundance of ABCA1 protein. Thus, introduction of CYP27A1 into cells stimulates cholesterol efflux and therefore may increase protection against atherosclerosis.