Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist

L-660,711 (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid is a potent and selective competitive inhibitor of [3H]leukotriene D4 binding in guinea pig (Ki value, 0.22 nM) and human (Ki value, 2.1 nM) lung membranes but is essentially inactive versus [3H]leukotriene C4 binding (IC50 value in guinea pig lung, 23 microM). Functionally it competitively antagonized contractions of guinea pig trachea and ileum induced by leukotriene (LT) D4 (respective pA2 values, 9.4 and 10.5) and LTE4 (respective pA2 values, 9.1 and 10.4) and contractions of human trachea induced by LTD4 (pA2 value, 8.5). L-660,711 (5.8 x 10(-8)M) antagonized contractions of guinea pig trachea induced by LTC4 in the absence (dose ratio = 28) but not in the presence of 45 mM L-serine borate (dose ratio less than 2). L-660,711 (1.9 x 10(-5)M) did not block contractions of guinea pig trachea induced by histamine, acetylcholine, 5-hydroxytryptamine, PGF2 alpha, U-44069, or PGD2. In the presence of atropine, mepyramine, and indomethacin, L-660,711 (1.9 x 10(-5)M) inhibited a small component of the response to antigen on guinea pig trachea but completely blocked anti-IgE-induced contractions of human trachea. L-660,711 (i.v.) antagonized bronchoconstriction induced in anesthetized guinea pigs by i.v. LTC4, LTD4, and LTE4 but did not block bronchoconstriction to arachidonic acid, U-44069, 5-hydroxytryptamine, histamine, or acetylcholine. Intraduodenal L-660,711 antagonized LTD4 (0.2-12.8 micrograms/kg)-induced bronchoconstriction in guinea pigs, and p.o. L-660,711 blocked LTD4- and Ascaris-induced bronchoconstriction in conscious squirrel monkeys and ovalbumin-induced bronchoconstriction in conscious sensitized rats treated with methysergide (3 micrograms/kg). The pharmacological profile of L-660,711 indicates that it is a potent, selective, orally active leukotriene receptor antagonist which is well suited to determine the role played by LTD4 and LTE4 in asthma and other pathophysiologic conditions.     

Metabolic and morphologic properties of single muscle fibers in the rat after spaceflight, Cosmos 1887

The adaptation of a slow (soleus, Sol) and a fast (medial gastrocnemius, MG) skeletal muscle to spaceflight was studied in five young male rats. The flight period was 12.5 days and the rats were killed approximately 48 h after returning to 1 g. Five other rats that were housed in cages similar to those used by the flight rats were maintained at 1 g for the same period of time to serve as ground-based controls. Fibers were classified as dark or light staining for myosin adenosine triphosphatase (ATPase). On the average, the fibers in the Sol of the flight rats atrophied twice as much as those in the MG. Further, the fibers located in the deep (close to the bone and having the highest percentage of light ATPase and high oxidative fibers in the muscle cross section) region of the MG atrophied more than the fibers located in the superficial (away from the bone and having the lowest percentage of light ATPase and high oxidative fibers in the muscle cross-section) region of the muscle. Based on quantitative histochemical assays of single muscle fibers, succinate dehydrogenase (SDH) activity per unit volume was unchanged in fibers of the Sol and MG. However, in the Sol, but not the MG, the total amount of SDH activity in a 10-microns-thick section of a fiber decreased significantly in response to spaceflight. Based on population distributions, it appears that the alpha-glycerophosphate dehydrogenase (GPD) activities were elevated in the dark ATPase fibers in the Sol, whereas the light fibers in the Sol and both fiber types in the MG did not appear to change. The ratio of GPD to SDH activities increased in the dark (but not light) fibers of the Sol and was unaffected in the MG. Immunohistochemical analyses indicate that approximately 40% of the fibers in the Sol of flight rats expressed a fast myosin heavy chain compared with 22% in control rats. Further, 31% of the fibers in the Sol of flight rats expressed both fast and slow myosin heavy chains compared with 8% in control rats. Immunohistochemical changes in the MG were minimal. These data suggest that the magnitude and direction of enzymatic activity and cell volume changes are dependent on the muscle, the region of the muscle, and the type of myosin expressed in the fibers. Further, the ability of fibers to maintain normal or even elevated activities per unit volume of some metabolic enzymes is remarkable considering the marked and rapid decrease in fiber volume.     

In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II.

Dystrophin is a cytoskeletal protein which is thought to play an important role in membrane physiology since its absence (due to gene deficiency) leads to the symptoms of Duchenne muscular dystrophy (DMD). Some disruption in the regulation of intracellular free Ca2+ levels could lead to DMD-like symptoms. In this study, calpains, which are very active calcium-dependent proteases, were examined for their capacity to hydrolyse dystrophin in vitro. The results show that calpains are able to split dystrophin and produce breakdown products of different sizes (the degree of cleavage being dependent on the incubation time with proteases). The time-course of protease degradation was examined by Western immunoblot using three polyclonal sera which were characterized as being specific to the central (residues 1173-1728) and two distal parts of the molecule ie specific to the N-terminal (residues 43-760) or the C-terminal (residues 3357-3660) extremities of the dystrophin molecule. The cleavage patterns of dystrophin showed an accumulation of some major protease-resistant fragments of high relative molecular mass (250-370 kDa). These observations demonstrate that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation. For instance, it is clear that calpains first give rise to large dystrophin products in which the C-terminal region is lacking. These observations suggest that dystrophin antibodies specific to the central domain of the molecule should be used to detect dystrophin for diagnostic purposes and before any conclusion as to the presence or absence of dystrophin can be deduced from results obtained using immunoanalyses of muscle biopsies.     

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.     

Anti-myosin heavy chain monoclonal antibodies reveal two IIB (fast) fiber subtypes

Indirect immunofluorescence analysis of different rat skeletal muscles using anti-myosin heavy chain (MHC) monoclonal antibodies (MAb) revealed the presence of two immunologically distinct kinds of fibers within the IIB fibers, histochemically identified by myosin ATPase staining. Some IIB fibers (designated here as IIB1) were unreactive with one anti-fast MHC MAb, whereas they did react with another anti-fast MHC MAb; other IIB fibers (designated here as IIB2) reacted with both anti-fast MAbs. Neither of the two IIB fiber subtypes was significantly reactive with a neonatal MHC MAb. The number of each IIB fiber subtype was age-dependent, at least in the plantaris muscle. IIB1 fibers were observed only in the superficial portion of the plantaris and gastrocnemius muscle. The ratio of IIB1:IIB2 fibers was about the same throughout the extensor digitorum longus and extraocular muscles. Therefore, the two kinds of IIB fibers here observed have a different myosin heavy chain content. On the basis of their specific immunoreactivities, we suggest that IIB1 fibers contain the previously described MHCB. IIB2 fibers contain either a unique new MHC isoform or a mixture of at least two MHC, possibly composed of the MHCB and either the previously described MHCA or a new MHC isoform.     

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.      

Cloned DNA probes distinguish endemic and exotic Entomophaga grylli fungal pathotype infections in grasshopper life stages

Entomophaga grylli is a fungal pathogen of grasshoppers and at least three pathotypes are recognized world-wide. Pathotypes 1 and 2 are endemic to North America while the Australian pathotype 3 had been released into two field sites in North Dakota between 1989 and 1991. Grasshoppers were collected over the summer at the field sites in 1992 and assessed for pathotype infection by cloned DNA probe analysis. The three most predominant grasshopper species that were infected ( Melanoplus sanguinipes, M. bivittatus and Camnula pellucida ) were assessed for pathotype infection with respect to their life stages (nymphal instars and adult males and females). Pathotype 1 predominantly infected grasshoppers in the subfamilies Oedipodinae and Gomphocerinae and pathotype 2 predominantly infected grasshoppers in the subfamily Melanoplinae. Early-instar M. sanguinipes and M. bivittatus had higher pathotype 2 infection frequencies, while late-instar and adult C. pellucida had higher pathotype 1 infection frequencies. Cross-infection by the pathotypes did occur in up to 3% of the individuals, on a per species basis, and primarily in later instar and adult grasshoppers. Pathotype 3 infections occurred in later instar and adults of the three grasshopper species. Infection of grasshoppers by E. grylli pathotypes is discussed with reference to the fungal life cycles.