Triacylglycerol and phospholipid fatty acids of the silverleaf whitefly: composition and biosynthesis

The identification and composition of the fatty acids of the major lipid classes (triacylglycerols and phospholipids) within Bemisia argentifolii Bellows and Perring (Homoptera: Aleyrodidae) nymphs were determined. Comparisons were made to fatty acids from the internal lipids of B. argentifolii adults. The fatty acids, as ester derivatives, were analyzed by capillary gas chromatography (CGC) and CGC-mass spectrometry (MS). All lipid classes contained variable distributions of eight fatty acids: the saturated fatty acids, myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0); the monounsaturated fatty acids, palmitoleic acid (16:1), oleic acid (18:1); the polyunsaturated fatty acids, linoleic acid (18:2), linolenic acid (18:3). Fourth instar nymphs had 5-10 times the quantities of fatty acids as compared to third instar nymphs and 1-3 times the quantities from adults. The fatty acid quantity differences between fourth and third instar nymphs were related to their size and weight differences. The percentage compositions for fatty acids from each lipid class were the same for the pooled groups of third and fourth instar nymphs. For nymphs and adults, triacylglycerols were the major source of fatty acids, with 18:1 and 16:0 acids as major components and the majority of the polyunsaturated fatty acids, 18:2 and 18:3 were present in the two phospholipid fractions, phosphatidylethanolamine and phosphatidylcholine. Evidence was obtained that whiteflies indeed synthesize linoleic acid and linolenic acid de novo: radiolabel from [2-(14)C] acetate was incorporated into 18:2 and 18:3 fatty acids of B. argentifolii adults and CGC-MS of pyrrolidide derivatives established double bonds in the Delta(9,12) and Delta(9,12,15) positions, respectively.     

3D-biohybrid systems: applications in drug screening

Biotechnology demands powerful methods for the functional characterisation and monitoring of molecular alterations in tissues in response to various stimuli. Currently, cellular biosensors provide information about cell and tissue internal transduction pathways. In this article, recent biosensor systems are briefly described and the use of 3D tissue aggregates as recognition elements is discussed. An example of an innovative approach for drug testing using 3D heart muscle aggregates, as well as tumor models, positioned in capillary systems for electrical potential recording and impedance measurement is described. The effectiveness of drugs and therapies can be tested and monitored in a short time using such biohybrid sensors.     

Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.     

Tumor progression despite efficient tumor antigen cross-presentation and effective "arming" of tumor antigen-specific CTL

To determine whether APC function or "arming" of CTL for lytic function are the points at which Ags from a nonimmunogenic tumor fail to induce an effective immune response, we established a murine tumor model that expressed intracellular OVA and selected a clone (cOVA-9) that remained susceptible to lysis by specific CD8(+) T cells throughout tumor growth. Viable cOVA-9 tumor cells grew in normal mice at a rate similar to the parental tumor, and vaccination with irradiated cOVA-9 cells did not induce protection against itself or the parental line, confirming its nonimmunogenic status. In vivo evaluation during tumor growth demonstrated persisting tumor Ag cross-presentation accompanied by the generation of potent, specific CTL which were detectable when tumors were barely palpable. Despite the presence of highly active CTL in the tumor-draining lymph nodes, there was no apparent lysis of tumor-associated APC. These data show that tumor-draining APC are not dysfunctional with regard to two crucial processes, in vivo tumor Ag cross-presentation and specific CTL arming, and that failure to prevent tumor growth is not in the induction phase, but in the effector phase and occurs within the tumor itself before the tumor matrix is established.     

Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a beta-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)-selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.     

Monosialoganglioside increases catalase activity in cerebral cortex of rats

Monosialoganglioside (GM1) is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. However, only a few studies have attempted to investigate the effects of GM1 on enzymatic antioxidant defenses of the brain. In the present study, we evaluate the effects of the systemic administration of GM1 on the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and on spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cerebral cortex of rats ex vivo. The effects of GM1 on CAT activity and spontaneous chemiluminescence in vitro were also determined.

Animals received two injections of GM1 (50 mg/kg, i.p.) or saline (0.85% NaCl, i.p.) spaced 24 h apart. Thirty minutes after the second injection the animals were sacrificed and enzyme activities and spontaneous chemiluminescence and TRAP were measured in cell-free homogenates. GM1 administration reduced spontaneous chemiluminescence and increased catalase activity ex vivo, but had no effect on TRAP, SOD or GSH-Px activities. GM1, at high concentrations, reduced CAT activity in vitro. We suggest that the antioxidant activity of GM1 ganglioside in the cerebral cortex may be due to an increased catalase activity.     

Intracellular multiplication of Legionella pneumophila depends on host cell amino acid transporter SLC1A5

The infectious agent of Legionnaires' disease, Legionella (L) pneumophila, multiplies intracellularly in eukaryotic cells. This study has been performed to explore the nutrient requirements of L. pneumophila during intracellular replication. In human monocytes, bacterial replication rate was reduced by 76% in defined medium lacking L-cysteine, L-glutamine or L-serine. SLC1A5 (hATB(0,+)), a neutral amino acid transporter, was upregulated in the host cells after infection with L. pneumophila. Inhibition of SLC1A5 by BCH, a competitive inhibitor of amino acid uptake as well as siRNA silencing of the slc1a5 gene blocked intracellular multiplication of L. pneumophila without compromising viability of host cells. These observations suggest that replication of L. pneumophila depends on the function of host cell SLC1A5.