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.     

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.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

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.     

Congruent population genetic structures and divergence histories in anther‐smut fungi and their host plants Silene italica and the Silene nutans species complex

Study of the congruence of population genetic structure between hosts and pathogens gives important insights into their shared phylogeographical and coevolutionary histories. We studied the population genetic structure of castrating anther‐smut fungi (genus Microbotryum) and of their host plants, the Silene nutans species complex, and the morphologically and genetically closely related Silene italica, which can be found in sympatry. Phylogeographical population genetic structure related to persistence in separate glacial refugia has been recently revealed in the S. nutans plant species complex across Western Europe, identifying several distinct lineages. We genotyped 171 associated plant–pathogen pairs of anther‐smut fungi and their host plant individuals using microsatellite markers and plant chloroplastic single nucleotide polymorphisms. We found clear differentiation between fungal populations parasitizing S. nutans and S. italica plants. The population genetic structure of fungal strains parasitizing the S. nutans plant species complex mirrored the host plant genetic structure, suggesting that the pathogen was isolated in glacial refugia together with its host and/or that it has specialized on the plant genetic lineages. Using random forest approximate Bayesian computation (ABC‐RF), we found that the divergence history of the fungal lineages on S. nutans was congruent with that previously inferred for the host plant and probably occurred with ancient but no recent gene flow. Genome sequences confirmed the genetic structure and the absence of recent gene flow between fungal genetic lineages. Our analyses of individual host–pathogen pairs contribute to a better understanding of co‐evolutionary histories between hosts and pathogens in natural ecosystems, in which such studies remain scarce.