Fatty acid composition of fat body and malpighian tubules of the tenebrionid beetle, Zophobas atratus: Significance in eicosanoid-mediated physiology

Total and phospholipid fatty acid composition of fat body and Malpighian tubules from two larval stages, pupae and adults, of Zophobas atratus were analyzed. Saturated and unsaturated C16 and C18 fatty acids were major components and varied by life stage and tissue source. Eicosanoid-precursor fatty acids, including 20:3n-6, 20:4n-6 and 20:5n-3, were present in low quantities and varied by life stage and tissue source. 20:3n-6 was always present in the lowest proportions, indicating that eicosanoids derived from 20:4n-6 and 20:5n-3 (the 2- and 3-series) are likely to be of greater physiological significance in this insect. Fatty acid composition of Z. atratus fat body and Malpighian tubules was independent of diet, suggesting that this insect controls its fatty acid composition to meet the needs of individual tissues and ontogenetic constraints.     

Removal of insect basal laminae using elastase

We have used the enzyme elastase to remove the basal lamina of epithelia from two insects: the upper Malpighian tubules of Rhodnius prolixus and imaginai discs of Drosophila melanogaster. Removal of the basal lamina was confirmed using scanning and transmission electron microscopy. Use of the technique on the Malphighian tubules of Rhodnius reveals for the first time the three-dimensional organization of the circumferential folds of the basal plasma membrane. Elastase is much more effective in removing the basal lamina than are the enzymes hyaluronidase, collagenase, and chymotrypsin, either alone or in combination. Following elastase treatment, cells of the Malpighian tubules dissociate with only mild mechanical agitation into single, viable cells. Treatment with elastase removes the basal laminae of imaginai discs of Drosophila and accelerates evagination as has been previously described for trypsin. To obtain single cell preparations from elastase-treated imaginai discs, mechanical stirring in Ringer low in Ca²⁺ was required. In addition to its usefulness in cell isolation, elastase treatment allows examination of the effect of removal of basal laminae on the physiology and development of insect epithelia.     

Extracellular purines from cells of seminiferous tubules

It has been long postulated that extracellular purines can modulate the function of the male reproductive system by interacting with different purinergic receptors of Sertoli and germinative cells. Many authors have described the biological changes induced by extracellular ATP and/or adenosine in these cells, and some hypothetical models for paracrine communication mediated by purines were proposed; however, the cellular source(s) of these molecules in seminiferous tubules remains unknown. In this study, we demonstrated for the first time that Sertoli cells are able to release ATP (0.3 nmol/mg protein) and adenosine (0.1 nmol/mg protein) in the extracellular medium, while germinative and myoid peritubular cells are able to secrete adenosine (0.02 and 0.37 nmol/mg protein, respectively). Indeed, all the three types of cells were able to release inosine at significant concentrations (about 0.4 nmol/mg protein). This differential secretion depending on the cellular type suggests that these molecules may be involved in the paracrine regulation and/or control of the maturation processes of these cells.     

Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2

The multidrug transporter MDR-1 P-glycoprotein (Pgp) has been recently pointed out as an important mechanism underlying chemical interaction between drugs and many commonly ingested substances, including grapefruit juice (GFJ). Modulation of intestinal Pgp dependent transport by GFJ may lead to changes in bioavailability of drugs that are substrates of Pgp itself, by affecting their presystemic clearance. Since other cellular sites expressing Pgp and devoted to drug disposition, like kidney proximal tubules, could be involved in these pharmacokinetic interactions, we investigated the effect of GFJ on the expression and activity of Pgp in the human immortalized tubular cell line HK-2. Two flavonoid compounds related to GFJ, kaempferol and naringenin, were also tested for their effects on HK-2 Pgp. HK-2 cells cultured for 4 days in the presence of GFJ, showed a dose-dependent decrease in Pgp immunoblottable amount as well as a decrease in MDR-1 mRNA level, as shown by western blot analysis and RT-PCR, respectively. Both kaempferol and naringenin were also able to significantly decrease Pgp immunoblottable amount. To test whether the downregulation of HK-2 Pgp due to GFJ exposition could influence the cell sensitivity to drugs that are transported by Pgp itself, HK-2 cells precultured with GFJ were exposed to scalar concentrations of Cyclosporin A or Vinblastine and cell viability examined 36 hours later. The cytotoxicity of both drugs was increased. The calcein-AM test in untreated cells showed that GFJ, kaempferol or naringenin inhibited Pgp activity. Downregulation of Pgp as well inhibition of its function by GFJ or its related components in tubular cells could have a role in changing disposition kinetics of some important therapeutic agents.     

Characterization of salicylate uptake across the basolateral membrane of the malpighian tubules of Drosophila melanogaster

The organic anion salicylate is a plant secondary metabolite that can protect plants against herbivores. Transport of salicylate across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster was studied using a radioisotope tracer technique. The uptake of [(14)C]salicylate by the Malpighian tubules was active, saturable and Na(+)-dependent; the maximum uptake rate (J(max)) and the half saturation concentration (K(t)) were 12.6 pmoltubule(-1)min(-1) and 30.7micromoll(-1), respectively. In contrast to organic anion transport by vertebrate renal tissues, salicylate uptake was not trans-stimulated by glutarate (0.01-1.0 mmoll(-1)) or cis-inhibited by high concentrations (5 mmoll(-1)) of various alpha-keto acids (glutaric acid, alpha-ketoglutaric acid, succinic acid, and citric acid). Changes in basolateral membrane potential or physiologically relevant changes in bathing saline pH did not affect the rate of [(14)C]salicylate uptake. Ring-structure monocarboxylic acids (benzoic acid, nicotinic acid, gentisic acid, unlabelled salicylic acid, alpha-cyano-4-hydroxycinnamic acid, probenecid, fluorescein, and P-aminohippuric acid) strongly inhibited [(14)C]salicylate uptake rate. In contrast, short-chain monocarboxylic acids had little (butyric acid) or no effect (lactic acid, pyruvic acid, and propionic acid). Our results suggest that salicylate uptake across the basolateral membrane of D. melanogaster Malpighian tubules is mediated by a non-electrogenic, alpha-cyano-4-hydroxycinnamic acid-sensitive, Na(+):salicylate cotransport system.     

Chlamydia trachomatis causes centrosomal defects resulting in chromosomal segregation abnormalities

Chlamydiae traffic along microtubules to the microtubule organizing center (MTOC) to establish an intracellular niche within the host cell. Trafficking to the MTOC is dynein dependent although the activating and cargo-linking function of the dynactin complex is supplanted by unknown chlamydial protein(s). We demonstrate that once localized to the MTOC, the chlamydial inclusion maintains a tight association with cellular centrosomes. This association is sustained through mitosis and leads to a significant increase in supernumerary centrosomes, abnormal spindle poles, and chromosomal segregation defects. Chlamydial infection thus can lead to chromosome instability in cells that recover from infection.