Identification of Amino Acid Residues Responsible for the Selectivity of Tadalafil Binding to Two Closely Related Phosphodiesterases,PDE5 and PDE6

The 11 families of the Class I cyclic nucleotide phosphodiesterases (PDEs) are critical for regulation of cyclic nucleotide signaling. PDE5 (important in regulating vascular smooth muscle contraction) and PDE6 (responsible for regulating visual transduction in vertebrate photoreceptors) are structurally similar but have several functional differences whose structural basis is poorly understood. Using evolutionary trace analysis and structural homology modeling in conjunction with site-directed mutagenesis, we have tested the hypothesis that class-specific differences between PDE5 and PDE6 account for the biochemical and pharmacological differences in the two enzyme families. Replacing human PDE5 residues in the M-loop region of the binding site for the PDE5-selective inhibitor tadalafil (Cialis®) with the corresponding class-specific cone PDE6 residues (P773E, I778V, E780L, F787W, E796V, D803P, L804M, N806D, I813L, S815K) reduces tadalafil binding affinity to levels characteristic of PDE6. These mutations fail to alter vardenafil (Levitra®) affinity for the active site. Class-specific differences in PDE5 versus cone PDE6 that contribute to the accelerated catalytic efficiency of PDE6 were identified but required heterologous expression of full-length PDE5 constructs. Introduction of PDE6 residues into the background of the PDE5 protein sequence often led to loss of catalytic activity and reduced protein solubility, supporting the idea that multiple structural elements of PDE6 are highly susceptible to misfolding during heterologous expression. This work validates the use of PDE5 as a template to identify functional differences between PDE5 and PDE6 that will accelerate efforts to develop the next generation of PDE5-selective inhibitors with fewer adverse side effects resulting from PDE6 inhibition.     

ICln, a novel integrin alphaIIbbeta3-associated protein, functionally regulates platelet activation

A critical role for the conserved alpha-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin alpha(IIb)beta(3). To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with alpha(IIb)beta(3) by surface plasmon resonance. The affinity of this interaction was 82.2 +/- 24.4 nm in a cell free assay. ICln co-immunoprecipitates with alpha(IIb)beta(3) in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 microm to 5 mm), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10-100 microm) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.     

Temperature dependence of rat diaphragm muscle contractility and fatigue

The diaphragm is a skeletal muscle of mixed fiber type that is unique in its requirement to maintain contractile function and fatigue resistance across a wide range of temperatures to sustain alveolar ventilation under conditions of hypo- or hyperthermia. The direct effect of temperature (15-41 degrees C) on rat diaphragm isometric contractility and fatigue was determined in vitro. As temperature decreased from 37 to 15 degrees C, contraction and relaxation times increased, and there was a left shift of the diaphragm's force-frequency curve, with decreased contractility at 41 and 15 degrees C. Fatigue was induced by 10 min of stimulation with 30 trains/min of 5 Hz at a train duration of 900 ms. Compared with 37 degrees C, fatigue resistance was enhanced at 25 degrees C, but no difference in fatigue indexes was evident at extreme hypothermia (15 degrees C) or hyperthermia (41 degrees C). Only when the fatigue program was adjusted to account for hypothermia-induced increases in tension-time indexes was fatigue resistance evident at 15 degrees C. These findings indicate that despite the diaphragm's unique location as a core structure, necessitating exposure to in vivo temperatures higher than found in limb muscle, the temperature dependence of rat diaphragm muscle contractility and fatigue is similar to that reported for limb muscle of mixed fiber type.     

The half-life of follicle-stimulating hormone in ovary-intact and ovariectomized booroola and control merino ewes

To determine whether the high ovulation rate of the Booroola Merino ewe could be explained by FSH metabolism we have tested the proposition that FSH may have a longer half-life in the plasma of Booroola Merino ewes than in control ewes. The half-life of plasma FSH was determined by removal of the pituitary gland, to abolish FSH secretion into the peripheral circulation, and monitoring by repeated blood sampling the subsequent decline in plasma FSH concentrations. The half-life of FSH was similar in Booroola (103 +/- 14 (s.e.m.) min, N = 8) and control (116 +/- 8 min, N = 9) ewes. However, when ewes that had been ovariectomized at least 6 months earlier were hypophysectomized, the half-life of FSH was increased from 110 + 8 min in ovary-intact ewes (N = 11) to 1101 +/- 49 min (N = 6) (P less than 0.001) with no difference between the two Merino strains. We conclude that changes in the circulating half-life of FSH do not account for the high fecundity of the Booroola but that ovariectomy can alter the half-life of FSH secreted by the pituitary gland.     

Howard Hughes Medical Institute and its role in genomic activities

Through its three operating programs, the Howard Hughes Medical Institute has supported a substantial amount of basic and clinical molecular research, has acted as a facilitator in initiating and funding a number of meetings, nationally and internationally, including work groups dealing with databases, and has supported several databases for gene mapping. Finally, HHMI is now playing a significant national role in the training and development of manpower through its support of fellowships, direct grants to colleges and universities, and support for major teaching resources such as Woods Hole Marine Biology and Cold Spring Harbor Laboratories.     

Induction of parturition in ewes with a single injection of oestradiol benzoate

A single $\text{im}$ injection of 20 mg of oestradiol benzoate administered to ewes at 142 – 148 days of gestation resulted in parturition in 65% and 84% of ewes within 48 and 96 hr respectively. The remaining 16% of treated ewes suffered a higher incidence of dystocia and lamb survival was poorer than in untreated ewes or the treated ewes which lambed within 96 hr of treatment.     

Investigation of the requirements for O-glycosylation by bovine submaxillary gland UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosamine transferase using synthetic peptide substrates

Peptides containing a triprolyl sequence carboxyl to a threonine residue can be O-glycosylated by a crude Triton x-100 extract of porcine submaxillary glands (Young, J. D., Tsuchiya, D., Sandlin, D. E., and Holroyde, M. J. (1979) Biochemistry 18, 4444-4448). In the present paper, we have studied the characteristics of the O-glycosylating enzyme, UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosamine transferase, from a membrane extract of bovine submaxillary glands using 11 synthetic peptide substrates in which the Thr-Pro-Pro-Pro was varied. The effect of these changes was measured by determining the apparent Km and Vmax values of the substrates. The studies thus far reveal: threonine cannot be glycosylated without a carboxyl triprolyl sequence; the alpha amino acid group of the threonine must be blocked; the nature of the group NH2-terminal to the threonine affects the kinetics of the reaction; and one residue can be between the threonyl and the triprolyl sequence. The triprolyl sequence in a protein may be an important signal for O-glycosylation.     

Scanning electron microscopy of echinoid podia

Summary Tube feet of the sea urchin Strongylocentrotus franciscanus were studied with the scanning electron microscope (SEM). By use of fractured preparations it was possible to obtain views of all components of the layered tube-foot wall.The outer epithelium was found to bear tufts of cilia possibly belonging to sensory cells. The nerve plexus was clearly revealed as being composed of bundles of varicose axons. The basal lamina, which covers the outer and inner surfaces of the connective tissue layer, was found to be a mechanically resistant and elastic membrane. The connective tissue appears as dense bundles of (collagen) fibers. The luminal epithelium (coelothelium) is a single layer of flagellated collar cells.There is no indication that the muscle fibers, which insert on the inner basal lamina of the connective tissue layer are innervated by axons from the basiepithelial nerve plexus.The results agree with previous conclusions concerning tube-foot structure based on transmission electron microscopy, and provide additional information, particularly with regard to the outer and inner epithelia.This investigation was supported by the Sonderforschungsbereich 138 of the Deutsche Forschungsgemeinschaft. The work was carried out at the Friday Harbor Laboratories of the University of Washington. The authors are indebted to the Director, Professor A.O.D. Willows for use of the facilities, and to Drs. Christopher Reed and Tom Schroeder for invaluable instruction and assistance