Circadian rhythm of serum and tissue lipids in fed and fasted rats

The oscillations of the free fatty acid concentration in the serum and white (epididymal) adipose tissue, of triglycerides in the serum and liver, of total serum, liver and adrenal cholesterol and of serum phospholipids were studied at 3-hour intervals for a period of 24 hours in fed male Wistar rats and in animals fasted for 24 hours (both adapted to an illumination regimen of 12 hours' light and 12 hours' darkness. The rhythm--studied by means of the cosinor analysis--was present in most of the given parameters; it was not recorded in the liver triglycerides and serum phospholipids of fasted rats and in the adrenal cholesterol of fed animals. Apart from the circadian rhythm, many parameters distinctly displayed an ultradian rhythm, mainly an approximately 12-hour period. In general, one day's starvation did not significantly affect the course of the circadian oscillations of the given indicators of rat lipid metabolism.     

Have the bloody cells gone to our heads?

Recent studies have shown that cells expressing neuronal antigens can be derived from a bone marrow transplant. A new report lends support to and extends these previous results by presenting compelling morphological evidence for the generation and integration of highly differentiated bone marrow-derived neurons.     

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.     

A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

Identification and design of a novel series of MGAT2 inhibitors

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to na?ve, p <0.01) in plasma triacylglycerol (TAG) concentration.     

A continuous fluorescence assay for lecithin cholesterol acyltransferase

A continuous fluorescence assay was adapted to the measurement of the phospholipase reaction of lecithin cholesterol acyltransferase (LCAT). The fluorescent phospholipid 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl phosphatidylcholine (C6-NBD-PC) in micelle form reacted with LCAT to yield NBD-caproic acid, resulting in up to 5-fold increases in fluorescence in 30 min. The reaction rates were optimal in mixtures containing 0.1 M NaCl and 4 mM beta-mercaptoethanol at 37 degrees C. Apolipoprotein A-I did not activate the enzyme and bovine serum albumin bound monomeric substrate and interfered with the fluorescence assay. Under similar reaction conditions, bee venom phospholipase A2 was almost 100-fold more reactive than LCAT.     

The role of endogenous estrogens in the maturation process of the bovine placenta

The plasma estrogen and progesterone concentrations of 19 pregnant cows (average duration of pregnancy 266.0 +/- 2.3 d at the start of the study) were determined daily from Day 6 pre partum to Day 1 post partum. Parturition was induced in all cows by administration of 10 mg i.m. flumethasone. Values were centered around the delivery date (Day 0) following either induced normal calving (n = 3) or surgical delivery (n = 16). In animals showing spontaneous expulsion of the fetal membranes (Group 1, n = 6) the average total estrogen concentration increased significantly from Day 6 until Day 1 before parturition (1329.2 +/- 317.9 to 3719.8 +/- 951.2 pg/ml in total estrogens). A marked decrease was observed on Day 1 post partum (459.4 +/- 344.2 pg/ml). In comparison with Group 1, animals showing either a delayed or partial expulsion of the fetal membranes, or in which the placenta could be withdrawn 16 h after calving (Group 2, n = 5), had consistently lower total estrogen concentrations between Day 6 (595.4 +/- 174.8 pg/ml) and Day 1 (1884.3 +/- 565.1 pg/ml) before parturition. The estrogen values of the cows with retained placenta (Group 3, n = 8) from Days 6 to 0 pre partum were significantly lower than those of Group 2. Total estrogen concentrations of the three groups 1 d post partum did not differ significantly. It is generally recognized that estrogens play an important role in the maturation process of the placentomes. Our investigation demonstrates that not only is the magnitude of the prepartum rise in estrogens of great influence of the maturation process but the duration of this rise is likewise important. These two factors are vital for a normal expulsion of the fetal membranes.     

Hydrogen peroxide cytotoxicity. Low-temperature enhancement by ascorbate or reduced lipoate.

The principal mechanism of H2O2 toxicity is thought to involve the generation of hydroxyl (HO.) radicals through its interactions with Fe2+ ions by the Fenton reaction. Of particular interest has been the demonstration by Ward, Blakely & Joner [(1985) Radiat. Res. 103, 383-392] that the cytotoxicity of H2O2 is diminished at low temperature. We have now examined this phenomenon further with a mammalian epithelial cell line (CNCMI-221). Resistance of these cells to 100 microM-H2O2 added extracellularly exhibits a transition in the temperature range between 27 degrees C and 22 degrees C. We have found that the low-temperature resistance to cytotoxic concentrations of H2O2 is abolished by preincubation of cells with reductants such as ascorbate or reduced lipoic acid. This implies that the low-temperature resistance to H2O2 cytotoxicity may be due to inhibition of cellular reductive processes. The restoration of the cytotoxic action of H2O2 at 4 degrees C by ascorbate is prevented by pre-exposure of cells to desferrioxamine. This is evidence that transition-metal ions (such as iron ions) are involved in the cytotoxicity and is consistent with a mechanism of cell damage that depends on the Fenton reaction and a metal ion in the reduced state. Restoration of H2O2 cytotoxicity at low temperature by ascorbate is consistent with the artificial production of an intracellular reducing environment that at normal temperatures is sustained by cellular metabolism.