Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bax and Bad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (West ern immunoblots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in control distal cells, Bcl-X-L and Bax had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bax, and Bcl-X-L, but not Bad, were endogenously expressed in control proximal cells. Bcl-X-L was significantly decreased in treated proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant increases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X-L in proximal cells, as well as translocation of Bcl-X-L protein to mitochondria within the surviving distal cells.

Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Taeniogonalos raymenti Carmean & Kimsey (Hymenoptera : Trigonalidae) reared as a hyperparasite of Sturmia convergens (Weidemann) (Diptera : Tachinidae), a primary parasite of Danaus plexippus (L.) (Lepidoptera : Nymphalidae)

Taeniogonalos raymenti is confirmed as a hyperparasitoid of the tachinid Sturmia convergens which parasitises larval Danaus plexippus. Trigonalids are indirect parasitoids and in this case we have direct evidence that wasp eggs must have been laid on the caterpillar's host plant. Asclepias fruticosa. before the secondary host, but not necessarily before the primary tachinid host, was present. Levels of hyperparasitism during our sampling period were very low at less than two percent.

Expression of asparagine synthetase in response to carbohydrate supply in model callus cultures and shoot tips of asparagus (Asparagus officinalis L.)

Sugar uptake and metabolism were studied in callus cultures and shoot tips of asparagus. Asparagus callus cultures were used to model senescence in shoot tips. Callus cultures absorbed glucose from a nutrient medium, and accumulated sucrose, glucose and fructose. This uptake of glucose by the callus cultures down-regulated expression of asparagine synthetase and beta -galactosidase transcripts that otherwise accumulated when sugar was withheld. When 80 mm-long asparagus shoots were excised from growing plants and placed in 2% and 8% sucrose solutions, endogenous concentrations of sucrose, glucose, fructose, UDPglucose, and glucose-6-phosphate declined in the 30mm-long meristematic tip regions. At the same time, asparagine and asparagine synthetase gene transcripts began to accumulate in these tips. When 10 mm-long asparagus shoot tips were placed on glucose- or fructose-containing agar, the tips accumulated sucrose, glucose and fructose, and asparagine accumulation and expression of asparagine synthetase were marginally reduced. We concluded that in callus cultures, asparagine synthetase expression was sugar regulated, but that sugar regulation was not as pronounced in asparagus shoot tips. This may be due in part to slower rates of sugar uptake into shoot tips and in part to compartmentation of sugars in the tips. We suggest that callus cultures are not a suitable model for metabolic studies in asparagus shoot tips.

Effects of moist chilling and solid matrix priming on germination of loblolly pine (Pinus taeda L.) seeds

Loblolly pine ( Pinus taeda L.) seeds from sources with a mild climate under maritime influence (North Carolina) required shorter moist chilling to achieve maximum germination vigor than seeds from sources with a harsher continental climate (Oklahoma). Solid matrix priming (SMP) for 6 d achieved as much as 60 d of moist chilling to improve rapidity, synchrony and completeness of germination for three of the four families studied. SMP after moist chilling increased the rapidity, synchrony and completeness of germination. The benefit of SMP was greatest for non-stratified seeds and the benefit decreased with length of moist chilling. In general, delaying planting for one week after SMP had minor effects on germination when seeds were kept in the SMP matrix at 4 degreesC. Delayed planting after SMP can increase germination rapidity and synchrony of seeds that have received long moist chilling and reduce the benefit of SMP in non-moist-chilled seeds.

Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L. (Platyhelminthes; Trematoda)

The complete nucleotide sequence of the mitochondrial (mt) DNA molecule of the liverfluke, Fasciola hepatica (phylum Platyhelminthes, class Trematoda, family Fasciolidae), was determined, It comprises 14462 bp, contains 12 protein-encoding, 2 ribosomal and 22 transfer RNA genes, and is the second complete flatworm (and the first trematode) mitochondrial sequence to be described in detail. All of the genes are transcribed from the same strand. Of the genes typically found in mitochondrial genomes of eumetazoans, only atp8 is absent. The nad4L and nad4 genes overlap by 40 nt. Most intergenic sequences are very short. Two larger non-coding regions are present. The longer one (817 nt) is located between trnG and cox3 and consists of 8 identical tandem repeats of 85 nt, rich in G and C, followed by 1 imperfect repeat. The shorter non-coding region (187 nt) exhibits no special features and is separated from the longer region by trnG. The gene arrangement resembles that of some other trematodes including the eastern Asian Schistosoma species (and cyclophyllidean cestode species) but it is strikingly different from that of the African schistosomes, represented by Schistosoma mansoni. The genetic code is as inferred previously for flatworms. Transfer RNA genes range in length from 58 to 70 nt, their products producing characteristic 'clover leaf' structures, except for tRNA(S-VON) and tRNA(S-AGN) lacking the DHU arm.

Effect of propionyl-L-carnitine on exercise performance in peripheral arterial disease

Background: Supplementation with propionyl-L-carnitine (PLC) may be of use in improving the exercise capacity of people with peripheral arterial disease. Methods: After a 2-wk exercise familiarization phase, seven subjects displaying intermittent claudication were studied over a 12-wk period consisting of three 4-wk phases, baseline (B), supplementation (S), and placebo (P). PLC was supplemented at 2 g(.)d(-1), and subjects were blinded to the order of supplementation. Unilateral calf strength and endurance were assessed weekly. Walking performance was assessed at the end of each phase using an incremental protocol, during which respiratory gases were collected. Results: Although there was not a significant increase in maximal walking time (similar to 14%) in the whole group, walking time improved to a greater extent than the individual baseline coefficient of variation in four of the seven subjects. The changes in walking performance were correlated with changes in the respiratory exchange ratio both at steady state (r = 0.59) and maximal exercise (r = 0.79). Muscle strength increased significantly from 695 +/- 198 N to 812 +/- 249 N by the end of S. Changes in calf strength from B to S were modestly related to changes in walking performance (r = 0.56). No improvements in calf endurance were detected throughout the study. Conclusions: These preliminary data suggest that, in addition to walking performance, muscle strength can be increased in PAD patients after 4 wk of supplementation with propionyl-L-carnitine.

High-performance L-band series and parallel switches using low-cost p-i-n diodes

Low-cost UHF-band p-i-n diodes are used to develop high-performance L-band series and parallel switches. To stop the rectification of large RF, signals, the diodes are biased at a large reverse-bias voltage. Parasitic elements of the diodes are tuned out using LC circuits in biasing circuits without increasing the size of the switches. (C) 2002 John Wiley Sons, Inc.