Inhibition of fast axonal transport in vitro by tetracaine: an increase in potency at alkaline pH, and no change in potency in calcium-depleted nerves

Some of the present in vitro experiments compare the degree of inhibition of fast axonal transport produced by tetracaine at neutral and at alkaline pH. In desheathed spinal nerves from bullfrog, 0.5 mM tetracaine reduced the quantity of [3H]leucine-labeled proteins which were transported to a ligature by 43% at pH 7.2 and by 96% at pH 8.2; separate experiments established that transport was not affected by the pH change in the absence of tetracaine. The relationship between pH and transport-blocking potency of tetracaine (pKa 8.2) is such that the local anesthetic is more potent when more uncharged form of the molecule is present; this may reflect the easier penetration across the axonal plasma membrane by the uncharged form of the tetracaine molecule. The axonal smooth endoplasmic reticulum has been attributed the function of a calcium reservoir, and it appeared possible that local anesthetics could block axonal transport by releasing calcium from this structure. However, the inhibition of transport produced by 1 mM tetracaine (pH 7.1) in sheathed nerves was approximately 80% both in nerves with a lower than normal calcium content (47% of normal) and in nerves with a normal calcium content; this result does not support the hypothesis that inhibition of axonal transport by local anesthetics is mediated by an increase in intracellular free Ca2+, but does not rule out the hypothesis either.     

EBP2 plays a key role in Epstein-Barr virus mitotic segregation and is regulated by aurora family kinases

Epstein-Barr virus (EBV) genomes persist indefinitely in latently infected human cells, in part due to their ability to stably segregate during cell division. This process is mediated by the viral EBNA1 protein, which tethers the viral episomes to the cellular mitotic chromosomes. We have previously identified a mitotic chromosomal protein, human EBNA1 binding protein 2 (hEBP2), which binds to EBNA1 and enables EBNA1 to partition EBV-based plasmids in Saccharomyces cerevisiae. Using an RNA silencing approach, we show that hEBP2 is essential for the proliferation of human cells and that repression of hEBP2 severely decreases the ability of EBNA1 and EBV-based plasmids to bind mitotic chromosomes. When expressed in yeast, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin as in human cells, and using yeast temperature-sensitive mutant strains, we found that the attachment of hEBP2 to mitotic chromosomes was dependent on the Ipl1 kinase. Both RNA silencing of the Ipl1 orthologue in human cells (Aurora B) and specific inhibition of the Aurora B kinase activity with a small molecule confirmed a role for this kinase in enabling hEBP2 binding to human mitotic chromosomes, suggesting that this kinase can regulate EBV segregation.     

Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.     

Dissociation of the inhibition of fast axonal transport by chlorimipramine from an effect on axonal microtubules

The effect of in vitro exposure of bullfrog spinal nerves to 0.2 mM chlorimipramine on the density of axonal microtubules was studied in an attempt to clarify the mechanism by which chlorimipramine inhibits fast axonal transport. A 17-h exposure to chlorimipramine reduced the density of microtubules in unmyelinated axons by only 18%; this microtubular loss does not reach the upper limit of the range of microtubule reduction associated with inhibition of fast axonal transport. A 23-h exposure to chlorimipramine, which had decreased microtubular density in unmyelinated axons by 40% in a previous study, did not decrease microtubular density in myelinated axons in the present study. These results rule out microtubular destruction as the mechanism responsible for inhibition of fast orthograde axonal transport by chlorimipramine, and greatly reduce the likelihood that microtubular destruction plays a significant role in the inhibition of fast retrograde transport by chlorimipramine.     

3beta-Hydroxysteroid dehydrogenase activity in human fetal membranes.

A 3beta-hydroxysteroid dehydrogenase (3betaHSD) was demonstrated in term human fetal membranes (chorion and amnion) with both dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3beta-hydroxy-5-pregnen-20-one as substrates, and the subcellular distribution substrate and nucleotide specificity of the enzyme was studied. In both membranes the microsomal fraction (particles which sedimented at 105,000 g after 90 min) had the highest specific activity. The chorion was more active than the amnion but the enzyme in both tissues had similar substrate and nucleotide specificity. NAD was the preferred cofactor, and pregnenolone was a better substrate than dehydroepiandrosterone in the presence of NAD. However, with NADP as cofactor both steroids were equally good substrates. When the 3beta-hydroxysteroid dehydrogenase activity of chorion microsomes was compared with that of placental microsomes, the specific activities were found to be of the same order of magnitude, and the substrate, nucleotide specificity and steroid binding properties were almost identical.     

Inhibition of Daxx-mediated apoptosis by heat shock protein 27

Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.     

Src-family kinase signaling,actin-mediated membrane trafficking and organellar dynamics in the control of cell fate: Lessons to be learned from the adenovirus E4orf4 death factor

Evidence has accumulated that there are different modes of regulated cell death, which share overlapping signaling pathways. Cytoskeletal-dependent inter-organellar communication as a result of protein and lipid trafficking in and out of organelles has emerged as a common, key issue in the regulation of cell death modalities. The movement of proteins and lipids between cell compartments is believed to relay death signals in part through modifications of organelles dynamics. Little is known, however, regarding how trafficking is integrated within stress signaling pathways directing organelle-specific remodeling events. In this review, we discuss emerging evidence supporting a role for regulated changes in actin dynamics and intracellular membrane flow. Based on recent findings using the adenovirus E4orf4 death factor as a probing tool to tackle the mechanistic underpinnings that control alternative modes of cell death, we propose the existence of multifunctional platforms at the endosome-Golgi interface regulated by SFK-signaling. These endosomal platforms could be mobilized during cell activation processes to reorganize cellular membranes and promote inter-organelle signaling.     

Oxidation of an oral [13C]glucose load at rest and prolonged exercise in trained and sedentary subjects

The purpose of this study was to compare the oxidation of[¹³C]glucose (100 g)ingested at rest or during exercise in six trained (TS) and sixsedentary (SS) male subjects. The oxidation of plasma glucose was alsocomputed from the volume of¹³CO₂and¹³C/¹²Cin plasma glucose to compute the oxidation rate of glucose released from the liver and from glycogen stores in periphery (mainly muscle glycogen stores during exercise). At rest, oxidative disposal of bothexogenous (8.3 ± 0.3 vs. 6.6 ± 0.8 g/h) and liver glucose (4.4 ± 0.5 vs. 2.6 ± 0.4 g/h) was higher in TS than in SS.This could contribute to the better glucose tolerance observed at rest in TS. During exercise, for the same absolute workload [140 ± 5 W: TS = 47 ± 2.5; SS = 68 ± 3 %maximal oxygen uptake(O_{2 max})], [¹³C]glucose oxidationwas higher in TS than in SS (39.0 ± 2.6 vs. 33.6 ± 1.2 g/h),whereas both liver glucose (16.8 ± 2.4 vs. 24.0 ± 1.8 g/h) and muscle glycogen oxidation (36.0 ± 3.0 vs. 51.0 ± 5.4 g/h) were lower. For the same relative workload (68 ± 3% O_{2 max}:TS = 3.13 ± 0.96; SS = 2.34 ± 0.60 lO₂/min), exogenous glucose(44.4 ± 1.8 vs. 33.6 ± 1.2 g/h) and muscle glycogen oxidation (73.8 ± 7.2 vs. 51.0 ± 5.4 g/h) were higher in TS. However,despite a higher energy expenditure in TS, liver glucose oxidation was similar in both groups (22.2 ± 3.0 vs. 24.0 ± 1.8 g/h). Thus exogenous glucose oxidation was selectively favored in TSduring exercise, reducing both liver glucose and muscle glycogen oxidation.

     

RNA localization in the cytoplasm

RNA localization in subcytoplasmic areas is a process known for more than twenty years, and more than a hundred RNAs have now been shown to be spatially regulated. In most cases, RNA localization is involved in cell polarity, either by reading spatial clues and translating them into a spatial regulation of gene expression, or more directly by controlling cytoskeletal polarity. In this review, the various functions of RNA localization will be presented, and by analyzing two examples, Ash1 mRNA in yeast and retroviral genomic RNAs in mammals, the reader will be taken step by step into the detailed mechanisms of this fascinating process.