G. Lesa

Long chain polyunsaturated fatty acids are required for efficient neurotransmission in C. elegans.

The complex lipid constituents of the eukaryotic plasma membrane are precisely controlled in a cell-type-specific manner, suggesting an important, but as yet, unknown cellular function. Neuronal membranes are enriched in long-chain polyunsaturated fatty acids (LC-PUFAs) and alterations in LC-PUFA metabolism cause debilitating neuronal pathologies. However, the physiological role of LC-PUFAs in neurons is unknown. We have characterized the neuronal phenotype of C. elegans mutants depleted of LC-PUFAs. The C. elegans genome encodes a single Δ6-desaturase gene (fat-3), an essential enzyme for LC-PUFA biosynthesis. Animals lacking fat-3 function do not synthesize LC-PUFAs and show movement and egg-laying abnormalities associated with neuronal impairment. Expression of functional fat-3 in neurons, or application of exogenous LC-PUFAs to adult animals rescues these defects. Pharmacological, ultrastructural and electrophysiological analyses demonstrate that fat-3 mutant animals are depleted of synaptic vesicles and release abnormally low levels of neurotransmitter at cholinergic and serotonergic neuromuscular junctions. These data indicate that LC-PUFAs are essential for efficient neurotransmission in C. elegans and may account for the clinical conditions associated with mis-regulation of LC-PUFAs in humans.

Chronic opiate agonists down-regulate prodynorphin gene expression in rat brain

The effects of long-term administration of opioid agonists on the regulation of prodynorphin gene expression in rat brain were investigated. Chronic intracerebroventricular treatment with the synthetic opioid agonist acting on the kappa receptor, U-50,488H, and the classic mu agonist morphine markedly decreased prodynorphin mRNA levels in hypothalamus, hippocampus and striatum of tolerant rats. Levels of ir-Dynorphin A remained unchanged except in two cases. Chronic exposure to opiates thus appears to induce modifications of the endogenous opioid system, as regards gene expression regulation.

The Amino-terminal Domain of the Golgi Protein Giantin Interacts Directly with the Vesicle-tethering Protein p115

Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat protomer I vesicles to Golgi membranes and the initial stacking of reforming cisternae. Here we show that the NH2-terminal 15% of Giantin suffices to bind p115 in vitro and in vivo and to block cell-free Golgi reassembly. Because Giantin is a long, rod-like protein anchored to the membrane by its extreme COOH terminus, these results support the idea of a long, flexible tether linking vesicles and cisternae.

Expression of ceramide glucosyltransferases, which are essential for glycosphingolipid synthesis, is only required in a small subset of C. elegans cells

Glycosphingolipids (GSLs) are glycosylated derivatives of ceramide in the lipid bilayer. Their ubiquitous distribution and complexity suggest that they have important functions, but what these are in vivo is still poorly understood. Here, we characterize the phenotype of Caenorhabditis elegans mutants with essentially no GSLs. The C. elegans genome encodes three ceramide glucosyltransferase (CGT) genes, which encode enzymes required for GSL biosynthesis. Animals lacking CGT do not synthesize GSLs, arrest growth at the first larval stage, and display defects in a subset of cells in their digestive tract; these defects impair larval feeding, resulting in a starvation-induced growth arrest. Restoring CGT function in these digestive tract cells – but not in a variety of other tissues – is sufficient to rescue the phenotypes associated with loss of CGT function. These unexpected findings suggest that GSLs are dispensable in most C. elegans cells, including those of the nervous system.

Skin levels of vasoactive intestinal polypeptide in atopic dermatitis

SummaryAtopic dermatitis (AD) can be exacerbated by various factors, including emotional stress, scratching and sweating. The aim of the present study was to evaluate the hypothesis that the inflammatory reaction in AD is also neurogenic. For this purpose, the levels of vasoactive intestinal polypeptide were measured radioimmunologically in whole-tissue homogenates of lesional skin of 13 patients with atopic dermatitis. Radioimmunoassay was performed using an antiserum, AH78, recognizing the carboxy-terminal fragment vasoactive intestinal polypeptide (22–28). Vasoactive intestinal polypeptide immunoreactivity was detected in relatively low amounts in control skin (0.428±0.08 pmol/g tissue), whereas a marked increase in the peptide was observed in lesional skin of patients with atopic dermatitis (5.62±1.25 pmol/g tissue). These results seem to suggest that vasoactive intestinal polypeptide could have a pathogenetic relevance in skin lesions of atopic dermatitis.

Long-term exposure to opioid antagonists up-regulates prodynorphin gene expression in rat brain

We investigated the effect of long-term administration of opioid antagonists on the regulation of prodynorphin gene expression in rat brain. Intracerebroventricular (i.c.v.) injections for seven days of nor-binaltorphimine (nor-BNI), the highly selective kappa opioid antagonist, naloxone and its longer acting analog naltrexone, both relatively selective antagonists for the mu opioid receptor, markedly raised prodynorphin mRNA levels in rat hypothalamus, hippocampus and striatum. Peptides, namely immunoreactive-dynorphin A (ir-dyn A), were unaffected after chronic treatment with all antagonists, in the same tissues. These results, taken together with our previous observations, suggest that chronic opioid antagonists, acting on kappa and mu opioid receptors, clearly up-regulate prodynorphin gene expression in discrete rat brain regions, activating its biosynthesis. Moreover, our data support the hypothesis that the endogenous opioid system plays a role in the mechanisms underlying the development of opiate tolerance.

Isolation of Caenorhabditis elegans gene knockouts by PCR screening of chemically mutagenized libraries

This protocol details methodologies to generate Caenorhabditis elegans deletion mutants by chemical mutagenesis and to detect them by PCR screening. Approximately, 600,000 worms are grown synchronously, mutagenized with ethyl methane sulfonate, divided in groups of 500 and allowed to self-fertilize for two generations. DNA is prepared from a fraction of each worm population, pooled into a 96-well plate, and screened by PCR with primers positioned 2.5–3.5 kb apart. Cultures containing deletion mutants are subdivided in small worm populations and tested again by PCR to identify positives. Single animals are then cloned from positive cultures, allowed to self-fertilize and identified by PCR genotyping. This method, which takes about a month, gives approximately a 50% chance of finding a deletion of interest larger than 500–600 bp. If a deletion cannot be found, the library can be pooled at lower complexity and screened for smaller deletions using an alternative PCR-based method.

A study of the enolization of some alkyl ketones in lyomesophases formed by alkylammonium surfactants

The kinetics of enolization of acetone, 3-pentanone, and 6-undecanone have been studied in lyotropic systems in the isotropic, nematic, and lamellar phases. The data, analysed in terms of spontaneous and acid-catalyzed rate constants, do not show any relevant influence of the physical nature of the phase. Effects are instead observed in relation to the length of the alkyl chains of the ketones and the type of the amphiphilic molecule. 6-undecanone has a surprising effect in stabilizing ordered mesophases.

Distribution and characterization of VIP-related peptides in the rat spinal cord

The possible existence in the rat spinal cord of a peptide related to VIP, VIP(22-28), has been evaluated. VIP contains paired basic aminoacid residues at which posttranslational cleavage of these peptides might occur. The lumbo-sacral region of rat spinal cord had the most VIP(22-28)-like immunoreactivity (ir-VIP(22-28]. Chromatographic analysis of spinal extracts showed that ir-VIP(22-28) consisted of two major peaks, one eluting as authentic VIP(1-28) and the other as VIP(22-28). HPLC confirmed these results, revealing the presence of intact VIP(1-28) and two or more less hydrophobic peptides, one of which corresponded to authentic VIP(22-28). The other two components found have not yet been identified. Further studies are necessary to provide information on the biological significance of VIP(22-28).