Cox-2 expression, PGE2 and cytokines production are inhibited by endogenously synthesized n-3 PUFAs in inflamed colon of fat-1 mice

There is great interest in the role of polyunsaturated fatty acids (PUFAs) in promoting (n-6 class) or inhibiting (n-3 class) inflammation. Mammalian cells are devoid of desaturase that converts n-6 to n-3 PUFAs. Consequently, essential n-3 fatty acids must be supplied with the diet. We have studied the effect of endogenously produced n-3 PUFAs on colitis development in fat-1 transgenic mice carrying the Caenorhabditis elegans fat-1 gene encoding n-3 desaturase. Colonic cell lipid profile was measured by capillary gas chromatography in fat-1 and wild-type (WT) littermates fed standard diet supplemented with 10% (w/w) safflower oil rich (76%) in n-6 polyunsaturated linoleic acid (LA). Experimental colitis was induced by administrating 3% dextran sodium sulphate (DSS). Colitis was scored by histopatological analysis. Cyclooxygenase-2 (Cox-2) expression was evaluated by real time polymerase chain reaction. Prostaglandin E₂ (PGE₂) levels and cytokine production were determined by enzyme and microsphere-based immunoassays, respectively. The n-6/n-3 PUFA ratios in colonic cells of fat-1 mice were markedly lower (9.83±2.62) compared to WT (54.5±9.24, P<.001). Results also showed an attenuation of colonic acute and chronic inflammation in fat-1 mice with significant decreases in PGE₂ production (P<.01) and Cox-2 expression (P<.01). High levels of colitis-induced proinflammatory cytokines, interleukin (IL)-18, IL-1α, IL-1β, IL-6, monocytes chemotactic proteins 1, 2 and 3 (MCP 1,2,3), matrix metalloproteinase 9 and tumor necrosis factor α (TNF-α) were down-regulated in DSS acutely and chronically treated fat-1 mice. The expression of fat-1 gene in the colon was associated with endogenous n-3 PUFAs production, decreased Cox-2 expression, increased PGE₂ and cytokine production.     

Structure activity relationship studies of carboxamido-biaryl ethers as opioid receptor antagonists (OpRAs). Part 2

A series of 6-bicycloaryloxynicotinamides were identified as opioid receptor antagonists at mu, kappa, and delta receptors. Compounds in the 6-(2,3,4,5-tetrahydro-1H-benzo[c]azepin-7-yloxy)nicotinamide scaffold exhibited potent in vitro functional antagonism at all three receptors.     

The mechanism of ran import into the nucleus by nuclear transport factor 2

The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-beta. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis in Caenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-beta-mediated protein import.     

Identification of resilin in the leg of cockroach, Periplaneta americana: confirmation by a simple method using pH dependence of UV fluorescence

We have examined the tarsus (foot) and tibial segments of the cockroach leg to identify structures that contain the elastic protein resilin. The presence of resilin was tested using the conventional criteria of fluorescent emission at 420 nm under UV illumination and histological staining of wholemount tissues by toluidine blue. We have also developed a simple method of confirming identification of resilin through changes in its fluorescence that occur with alteration of pH of the surrounding medium. Using a commonly available excitation filter that only passes light at >330 nm, we found that the emission was present at neutral pH but was eliminated at low pH. It then reversibly reappeared when medium of higher pH was restored. This effect is attributable to a known shift in the absorption maximum of amino acids of resilin that occurs in acidic media (from 330 to 285 nm). The accuracy of this method of identification was confirmed by examination of ligaments of the wing hinge, which has previously been shown to contain resilin in a number of insects. Using these techniques, we have identified resilin in association with ligaments at the tibio-tarsal joint and in the articulation between the fourth and fifth tarsal segments of the leg. The anatomical arrangement of these ligaments suggests that they could aid in the generation of leg movements during walking by functioning as elastic antagonists to the actions of leg muscles. The method of identification we have devised could readily be applied to aid in the localization of resilin in other animals.     

Towards a practical fail-safe system of managing poikilothermic vertebrate cell lines in culture

Summary A series of 13 integrated precepts is used in a system for managing continuous laboratory propagation of poikilothermic vertebrate cell lines. The precepts are listed in sequence of application and discussed.     

Infectious Pancreatic Necrosis Virus: Lyophilization and Subsequent Stability in Storage at 4 C

Infectious pancreatic necrosis virus (ATCC strain VR # 299) was lyophilized with different additives and stored at 4 C. Ampoules were assayed at 2 days, at 1, 3, and 6 months, and at 1, 3, and 4 years. Processing losses were least and subsequent maintenance of infectivity was best in skim milk, lactalbumin hydrolysate, and lactose. Quantitative data are given in tabular form.