Differential regulation of calmodulin-dependent and -independent cyclic AMP phosphodiesterases from oviduct by fatty acids.

Regulation of calmodulin-independent and -dependent cAMP phosphodiesterases from quail oviduct by various fatty acids was studied. The calmodulin-independent form was slightly activated by low concentrations (20 microM) of oleic, linoleic and arachidonic acid, higher concentrations were inhibitory. The basal activity of the calmodulin-dependent form was activated by linoleic acid and to a lesser extent by arachidonic acid at low concentrations and inhibited by higher concentrations of the two fatty acids. In contrast, arachidonic acid was a potent reversible inhibitor of calmodulin in the activation of this enzyme (IC50: 20 microM) whereas linoleic acid was inactive from 10 to 150 microM. The present results strongly suggest that the differential regulation of cAMP phosphodiesterases by these fatty acids could profoundly influence the level of cAMP in the oviduct and thus its subsequent effects.     

Caffeine or melatonin effects on sleep and sleepiness after rapid eastward transmeridian travel.

We measured the effects of slow-release caffeine (SRC) and melatonin (Mlt) on sleep and daytime sleepiness after a seven-time zone eastbound flight. In a double-blind, randomized, placebo-controlled study, each of three groups of nine subjects was given either 300 mg SRC on recovery day 1 (D1) to D5 (0800) or 5 mg Mlt on preflight D-1 (1700), flight day D0 (1600), and from D1 to D3 (2300), or placebo (Pbo) at the same times. Nighttime sleep was evaluated by polysomnography and daytime sleepiness from measurements of sleep latencies and continuous wrist actigraphy. Compared with baseline, we found a significant rebound of slow-wave sleep on night 1 (N1) to N2 under Pbo and Mlt and a significant decrease in rapid eye movement sleep on N1 (Pbo) and N1-N3 (Mlt). Sleepiness was objectively increased under Pbo (D1-D6) and Mlt (D1-D3). SRC reduced sleepiness but also tended to affect sleep quality until the last drug day. In conclusion, both drugs have positive effects on some jet lag symptoms after an eastbound flight: SRC on daytime sleepiness, and Mlt on sleep.     

Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT protein-dependent extraction of ceramides from the endoplasmic reticulum

To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.     

Differential membrane packing of stereoisomers of bis(monoacylglycero)phosphate

Bis(monoacylglycero)phosphate (BMP) reveals an unusual sn-1,sn-1' stereoconfiguration of glycerophosphate. We synthesized sn-(3-myristoyl-2-hydroxy)glycerol-1-phospho-sn-1'-(3'-myristoyl-2'-hydroxy)glycerol (1,1'-DMBMP) and characterized the thermotropic phase behavior and membrane structure, in comparison with those of the corresponding sn-3:sn-1' stereoisomer (3,1'-DMBMP), by means of differential scanning calorimetry (DSC), small- and wide-angle X-ray scattering (SAXS and WAXS, respectively), pressure-area (pi-A) isotherms, epifluorescence microscopy of monolayers, and molecular dynamics (MD) simulations. In DSC, these lipids exhibited weakly energetic broad peaks with an onset temperature of 9 degrees C for 1,1'-DMBMP and 18 degrees C for 3,1'-DMBMP. In addition, a highly cooperative, strongly energetic transition peak was observed at approximately 40 degrees C for 1,1'-DMBMP and approximately 42 degrees C for 3,1'-DMBMP. These results are supported by the observation that 1,1'-DMBMP exhibited a larger phase transition pressure (pi(c)) than 3,1'-DMBMP. Small- and wide-angle X-ray scattering measurements identified these small and large energetic transitions as a quasi-crystalline (L(c1))-quasi-crystalline with different tilt angle (L(c2)) phase transition and an L(c2)-L(alpha) main phase transition, respectively. X-ray measurements also revealed that these DMBMPs undergo an unbinding at the main phase transition temperature. The MD simulations estimated stronger hydrogen bonding formation in the 3,1'-DMBMP membrane than in 1,1'-DMBMP, supporting the experimental data.     

The origin and evolution of <Emphasis Type="Italic">ARGFX</Emphasis>homeobox loci in mammalian radiation

Background  

Many homeobox genes show remarkable conservation between divergent animal phyla. In contrast, the ARGFX (Arginine-fifty homeobox) homeobox locus was identified in the human genome but is not present in mouse or invertebrates. Here we ask when and how this locus originated and examine its pattern of molecular evolution.     

Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets

12-Hydroperoxy-eicosatetraenoic acid (12-HpETE), the main hydroperoxide formed in platelets from arachidonic acid (AA) by 12-lipoxygenase, has been shown to increase the sensitivity of platelets to agonists resulting in increased aggregation. The aim of the present study was to determine the direct effect of low concentrations of 12-HpETE on the signaling pathways leading to AA release from membrane phospholipids and thromboxane A2 (TxA2) formation. Exogenous 12-HpETE activated platelet p38 mitogen-activated protein kinase (p38 MAPK), as assessed by its phosphorylation, at a concentration as low as 100 nM and was much more potent than hydrogen peroxide. Moreover, the incubation of platelets with 100 nM 12-HpETE for 2 min led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). It was associated with a significant decrease in the concentration of AA esterified in phospholipids and an increased concentration of thromboxane B2, the stable catabolite of TxA2. Additionally, decreasing glutathione peroxidase activity pharmacologically favored endogenous 12-HpETE formation and led to an increase in phosphorylated p38 MAPK, while a thiol-reducing agent such as N-acetyl-cysteine fully prevented it. Finally, significant activation of p38 MAPK was also observed in platelets from type 2 diabetic patients with mild hyperglycemia. In conclusion, our data provide a new insight into the mechanism of 12-HpETE-induced platelet priming, suggesting that hydroperoxide-induced p38 MAPK activation could play a relevant role in the exacerbated platelet activation associated with oxidative stress as found in diabetes.     

Formation, Metabolism, and Action of Hepoxilin A3in the Rat Pineal Gland

Abstract: The present study was undertaken to investigate the possible formation of hepoxilin A₃ in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 μM) led to the appearance of hepoxilin A₃ (HxA₃) analyzed as its stable trihydroxy derivative, trioxilin A₃ by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA₃ is estimated to be 1.43 ± 0.66 ng//μg of protein. This amount is not modified when the tissue is boiled (2.07 ± 0.66 ng/μg of protein). However, the formation of this compound was stimulated to 21.26 ±5.82 ng/μg of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 /μg) resulted in a partial blockade of hepoxilin formation. Using [1-¹⁴C] H×A₃, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA₃ into trioxilin A₃. This hydrolysis was inhibited by 1 μmol/L of 3, 3, 3-trichloropropene-1, 2-oxide. In a separate study, HxA₃ in the presence of 3, 3, 3-trichloropropene-1, 2-oxide to block the hydrolysis of HxA₃ decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5′-N-ethylcarboxamidoadenosine, an A₁/A₂ adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (?88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA₃ in the rat pineal gland.     

Age-related changes in linoleic acid bioconversion by isolated hepatocytes from spontaneously hypertensive and normotensive rats

This study points out the hepatocyte interconversion of the linoleic acid family during hypertension. Hepatocyte 6 desaturase activity was higher in 1 month-old spontaneously hypertensive rats than in normotensive controls. A similar tendency was observed in 6 month-old SHR. 5 desaturase activity was higher only in 1 month-old spontaneously hypertensive rats as compared to controls. Desaturase activities were particularly high at the age of 6 months. The hepatocyte fatty acid composition showed an impairment of n-6 polyunsaturated fatty acid metabolism in spontaneously hypertensive animals. Changes were greater in the young prehypertensive rats than in adults. A storage of n-3 long chain fatty acids is remarkable in adult hypertensive rats, suggesting an alteration in peroxisomal oxidation. Such modifications may be related to the prostaglandin precursors availability to peripheral tissues such as kidney.