Critical considerations in the development of an assay for sulfidopeptide leukotrienes in plasma

A sensitive and specific assay has been developed for measurement of total sulfidopeptide leukotrienes (LT) in plasma. LTC4 and LTD4 in plasma are converted to LTE4 which is then extracted by C18 Sep-Pak binding and elution. Total LTE4 is resolved by reverse phase high performance liquid chromatography (RP-HPLC) and quantitated by radioimmunoassay (RIA). A [3H]LTE4 internal standard is added to the starting plasma sample to allow overall recovery to be calculated and to define the fractions from RP-HPLC to be assayed for LTE4-like immunoreactivity. The correlation between the measured increase in LTE4 concentration after addition of incremental amounts of LTC4 and LTE4 to plasma was 0.989 and 0.978, respectively, with slopes of 1.05 and 1.11. Addition of 51 pg/ml LTE4 to 5 ml plasma was detectable; the measured increase was 48 +/- 12 pg/ml (mean +/- SE, n = 7). The intra-assay coefficient of variation for 341 pg/ml of added LTC4 was 3.2% (n = 6). Sulfidopeptide leukotrienes could not be detected in blood samples taken from 12 normal volunteers in whom the theoretical detection limit, calculated from the sensitivity of the RIA, the overall recovery of LTE4, and the volume of plasma extracted, was 83 +/- 4 pg LTE4/ml plasma (0.19 +/- 0.01 pmol sulfidopeptide leukotriene/ml plasma; mean +/- SE).     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

The identification of a distinct export step following the biosynthesis of leukotriene C4 by human eosinophils

We have examined the requirements for the export of leukotriene C4 (LTC4) from cultured human eosinophils. To define saturability and kinetics of LTC4 export, eosinophils were interacted with leukotriene A4 (LTA4) at 37 degrees C, and the methanolic extracts of the cell-associated and extracellular compartments were then analyzed for LTC4 content by reverse phase high performance liquid chromatography with on-line monitoring of absorbance at 280 nm. When LTA4 was added at concentrations from 0 to 100 microM for 10 min at 37 degrees C, the amount of LTC4 released extracellularly became constant at an LTA4 concentration of 7.5 microM or greater even though the amount of intracellular LTC4 continued to increase. When eosinophils were incubated with 50 microM LTA4 for 0-60 min at 37 degrees C and then held at 0 degrees C for the remainder of the 60-min interval, 54.2 and 77.3% (n = 3), respectively, of the total LTC4 was released extracellularly after 15 and 30 min of incubation at 37 degrees C. Eosinophils incubated with 50 microM LTA4 at 0 degrees C for 1 h synthesized 290 pmol of LTC4 (n = 3) which was approximately half-maximal, all of which was retained intracellularly. We utilized the time and temperature dependence of LTC4 export to preload eosinophils with both LTC4 and leukotriene C5 (LTC5) by sequentially supplying them with specific substrates. With increasing concentrations of intracellular LTC5, there was dose-dependent inhibition of the subsequent release of LTC4 at 37 degrees C, with the sum of the released glutathionyl leukotrienes remaining constant. In addition, only minimal competition for LTC4 release occurred when cells were preloaded with both LTC4 and the conjugate of 1-chloro-2,4-dinitrobenzene and reduced glutathione, S-(dinitrophenyl)glutathione. The criteria of saturability, time dependence of LTC4 release at 37 degrees C, competition of LTC4 with LTC5 for release, and the inhibition of LTC4 release at 0 degrees C establish the export of LTC4 from cells as a novel and specific biochemical step distinct from both LTA4 uptake and the conjugation of LTA4 with reduced glutathione by LTC4 synthase to form LTC4.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes

Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.