A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.     

Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.     

Cis effect oflacZ sequences in transgenic mice

Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving theEscherichia coli -galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression inHMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of doubleHMG-lacZ andHMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that thelacZ sequence might interfere negatively with the expression of the adjacentHMG-cat transgene.     

Alterations in plasma-membrane functions after poliovirus infection

After exposure of HeLa cells to poliovirus there is a rapid decline (within minutes) in fluorescence polarization of DPH (1,6-diphenyl-1,3,5-hexatriene). Within one hour after infection the (Na+/K+)ATPase activity of an isolated plasma-membrane-rich fraction is enhanced, the cell volume decreases, and the intracellular concentration of a potent low-molecular-weight inhibitor of host protein synthesis increases.     

A Bayesian Interpretation of the Particle Swarm Optimization and Its Kernel Extension

Particle swarm optimization is a popular method for solving difficult optimization problems. There have been attempts to formulate the method in formal probabilistic or stochastic terms (e.g. bare bones particle swarm) with the aim to achieve more generality and explain the practical behavior of the method. Here we present a Bayesian interpretation of the particle swarm optimization. This interpretation provides a formal framework for incorporation of prior knowledge about the problem that is being solved. Furthermore, it also allows to extend the particle optimization method through the use of kernel functions that represent the intermediary transformation of the data into a different space where the optimization problem is expected to be easier to be resolved–such transformation can be seen as a form of prior knowledge about the nature of the optimization problem. We derive from the general Bayesian formulation the commonly used particle swarm methods as particular cases.     

Circadian rhythm does not affect responses to chemical cues in the red swamp crayfish,Procambarus clarkii

Studies of crayfish chemical ecology have been conducted in both day and night conditions. This variation may hinder the comparison of data among studies, if the responses by crayfish to chemical cues are dependent upon the time at which the cues are encountered. We tested the hypothesis that responses to chemical cues are dependent on observation time using the red swamp crayfish, Procambarus clarkii. Procambarus clarkii is known to exhibit a light-regulated circadian rhythm, with nocturnal activity peaks. Habitat use differed significantly between non-stimulated periods and periods of exposure to a food stimulus, but no effects of photoperiod (normal vs. reversed) or laboratory conditions (dark vs. light) were observed. The results suggest that, all else being equal, (1) studies of crayfish chemical ecology can be successfully conducted in a variety of experimental conditions, and (2) previous studies conducted at various times of the day should have comparable results.     

Caenorhabditis elegans as a model for intracellular pathogen infection

The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole‐animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal‐related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection.     

Roux-en-Y Gastric Bypass Increases Intravenous Ethanol Self-Administration in Dietary Obese Rats

Roux-en-Y gastric bypass surgery (RYGB) is an effective treatment for severe obesity. Clinical studies however have reported susceptibility to increased alcohol use after RYGB, and preclinical studies have shown increased alcohol intake in obese rats after RYGB. This could reflect a direct enhancement of alcohol’s rewarding effects in the brain or an indirect effect due to increased alcohol absorption after RGYB. To rule out the contribution that changes in alcohol absorption have on its rewarding effects, here we assessed the effects of RYGB on intravenously (IV) administered ethanol (1%). For this purpose, high fat (60% kcal from fat) diet-induced obese male Sprague Dawley rats were tested ∼2 months after RYGB or sham surgery (SHAM) using both fixed and progressive ratio schedules of reinforcement to evaluate if RGYB modified the reinforcing effects of IV ethanol. Compared to SHAM, RYGB rats made significantly more active spout responses to earn IV ethanol during the fixed ratio schedule, and achieved higher breakpoints during the progressive ratio schedule. Although additional studies are needed, our results provide preliminary evidence that RYGB increases the rewarding effects of alcohol independent of its effects on alcohol absorption.