Low-affinity binding sites for 1,4-dihydropyridines in skeletal muscle transverse tubule membranes revealed by changes in the fluorescence of felodipine.

The fluorescence changes accompanying the binding of the fluorescent calcium channel antagonist, felodipine, to transverse tubule membranes from rabbit skeletal muscle have been used to characterize low-affinity binding sites for 1,4-dihydropyridine derivatives in these preparations. In competition experiments, felodipine inhibited the high-affinity binding of (+)-[3H]PN200-110 to transverse tubule membranes with an apparent Ki of 5 +/- 2 nM. Binding of felodipine to additional low-affinity sites resulted in a large, saturable (Kd = 6 +/- 2 microM) increase in its fluorescence which could be excited either directly (380 nm) or indirectly via energy transfer from membrane protein (290 nm). The observed fluorescence enhancement was competitively inhibited by other 1,4-dihydropyridines with inhibition constants of 3-21 microM but was unaffected by the structurally unrelated calcium channel antagonists, diltiazem and verapamil, or by Ca2+, Cd2+, and La3+. Both high- and low-affinity binding sites appear to be localized in the transverse tubular system, since the magnitude of the observed fluorescence enhancement was higher in these membranes than in microsomal preparations and was directly proportional to the density of high-affinity sites for (+)-[3H]PN200-110. Furthermore, both high- and low-affinity sites appear to be conformationally coupled since, over the same concentration range that the fluorescence changes were observed, felodipine accelerated the rate of dissociation of [3H]PN200-110 previously bound to its high-affinity sites. Similar behavior has previously been reported for other 1,4-dihydropyridines [Dunn, S. M. J., & Bladen, C. (1991) Biochemistry 30, 5716-5721].(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray crystallographic analysis of inhibition of endothiapepsin by cyclohexyl renin inhibitors.

The crystal structures of endothiapepsin, a fungal aspartic proteinase (EC 3.4.23.6), cocrystallized with two oligopeptide renin inhibitors, PD125967 and PD125754, have been determined at 2.0-A resolution and refined to R-factors of 0.143 and 0.153, respectively. These inhibitors, which are of the hydroxyethylene and statine types, respectively, possess a cyclohexylalanine side chain at P1 and have interesting functionalities at the P3 position which, until now, have not been subjected to crystallographic analysis. PD125967 has a bis(1-naphthylmethyl)acetyl residue at P3, and PD125754 possesses a hydroxyethylene analogue of the P3-P2 peptide bond for proteolytic stability. The structures reveal that the S3 pocket accommodates one naphthyl ring with conformational changes of the Asp 77 and Asp 114 side chains, the other naphthyl group residing in the S4 region. The P3-P2 hydroxyethylene analogue of PD125754 forms a hydrogen bond with the NH of Thr 219, thereby making the same interaction with the enzyme as the equivalent peptide groups of all inhibitors studied so far. The absence of side chains at the P2 and P1' positions of this inhibitor allows water molecules to occupy the respective pockets in the complex. The relative potencies of PD125967 and PD125754 for endothiapepsin are consistent with the changes in solvent-accessible area which take place on inhibitor binding.     

The selectivity of statine-based inhibitors against various human aspartic proteinases.

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1' positions. The residues occupying the peripheral sub-sites (P4 to P3') were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.     

Does leukotriene C4 mediate hypoxic vasoconstriction in isolated ferret lungs?

To evaluate leukotriene (LT) C4 as a mediator of hypoxic pulmonary vasoconstriction, we examined the effects of FPL55712, a putative LT antagonist, and indomethacin, a cyclooxygenase inhibitor, on vasopressor responses to LTC4 and hypoxia (inspired O2 tension = 25 Torr) in isolated ferret lungs perfused with a constant flow (50 ml.kg-1.min-1). Pulmonary arterial injections of LTC4 caused dose-related increases in pulmonary arterial pressure during perfusion with physiological salt solution containing Ficoll (4 g/dl). FPL55712 caused concentration-related inhibition of the pressor response to LTC4 (0.6 micrograms). Although 10 micrograms/ml FPL55712 inhibited the LTC4 pressor response by 61%, it did not alter the response to hypoxia. At 100 microgram/ml, FPL55712 inhibited the responses to LTC4 and hypoxia by 73 and 71%, respectively, but also attenuated the vasoconstrictor responses to prostaglandin F2 alpha (78% at 8 micrograms), phenylephrine (68% at 100 micrograms), and KCl (51% at 40 mM). At 0.5 microgram/ml, indomethacin significantly attenuated the pressor response to arachidonic acid but did not alter responses to LTC4 or hypoxia. These results suggest that in isolated ferret lungs 1) the vasoconstrictor response to LTC4 did not depend on release of cyclooxygenase products and 2) LTC4 did not mediate hypoxic vasoconstriction.     

Spatial Patterns of Entomopathogenic Nematodes in Microcosms: Implications for Laboratory Experiments

Laboratory microcosms were used to: i) measure the effects of soil moisture on survival of Steinernema riobravis and ii) investigate the suitability of using microcosms to study motility and survival of these nematodes. Nematodes recovered from soil contained in petri dishes declined by more than 95% during 7 days, whereas nematodes recovered from the inner surfaces of dishes increased 35-fold. After 7 days in dishes, >20 times as many nematodes were recovered from dish surfaces than from soil. Nematodes exhibited a negative geotropism; greater numbers of nematodes were recovered from the lid surfaces than from the surfaces of dishes. Survivorship of nematodes in soil in plastic centrifuge tubes was somewhat greater than in petri dishes, and fewer nematodes ascended above the soil line in tubes than dishes. Downward migration of nematodes was inversely related to soil column diameter, possibly due to relatively unimpeded movement along container surfaces. An assay was developed by which nematodes were rinsed from the inner surfaces of centrifuge tubes into the soil. The resulting slurry was then processed on Baermann trays to recover motile nematodes. Nematode survival in soil in centrifuge tubes was higher at soil moistures between 2-4% than at lower (0.5-1.0%) and higher (4.0-12.0%) moisture levels. Survival of S. riobravis may be enhanced by quiescence induced by moisture deficits.     

Biology ofPterolonche inspersa (Lep.: Pterolonchidae), a biological control agent forCentaurea diffusa andC. maculosa in the United States

The moth,Pterolonche inspersa (Staudinger) (Lepidoptera: Pterolonchidae), is widely distributed in southern Europe, north Africa, Turkey and the former Soviet Union. It occurs in both thick and scattered stands of knapweeds in disturbed sites, usually on sandy and/or stony soil. Larvae bore in the roots of diffuse and spotted knapweeds (Centaurea diffusa De Lamarck andC. maculosa De Lamarck). There is one generation per year in northern Greece, and larvae feed in the roots for about 11 months during the growing season (August–September, to the following July–August). In the laboratory garden, emergence took place between the second half of July and the end of August, with peak emergence during mid August. In the field, adults were observed from early to late July. Female moths oviposited on rosettes during the first ten days of July and continued through the end of July. Eggs were laid singly or in groups of five or six, firmly attached to the leaves of the host plant. In the laboratory, females mated within 24 hours of emergence and the preoviposition period lasted 2.6±0.8 days. The oviposition period lasted 7.4±2.2 days and the average number of eggs per female was 142.2±59.2. The incubation period was 12±4.7 days; the pupal stage lasted 14.7±2.4 days; and females lived 15.8±2.4 days, while males lived 10.7±1.4 days. First instar larvae failed to survive on economically important Compositae in the generaCynara L.,Helianthus L.,Zinnia L. andCalendula L. (Dunnet al., 1989).