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Zinc phosphodiesterase (ZiPD) is a member of the metallo-(beta)-lactamase family with a binuclear zinc-binding site. The kinetic analysis of mutants provides unexpected insights into the reaction mechanism of ZiPD.

The three-dimensional structures of catalytic domain of human PDE3B in complex with a generic PDE inhibitor and a novel PDE3 selective inhibitor are presented. These structures provide the molecular basis for inhibitor specificity, and can supply a valid platform for the design of improved compounds.

Thg1p is identified by a survey of a genomic collection of yeast GST-ORF fusion proteins for addition of [alpha minus (super 32) P] GTP to tRNA (super His). Thg1p can catalyze phosphodiester bond formation at the 5' end of RNA in a 3'-5' direction.

Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide--gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:1147-161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by [Ca.sup.2+]-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced [Ca.sup.2+] influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the [K.sub.I] of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high [K.sub.m] for cAMP, whereas PDE type I has a low [K.sub.m] for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability. KEY WORDS: adenylyl cyclase * G-protein signaling * calcium influx * GH4 pituitary cells * biosensors

Phosphodiesterase 11A (PDE11A) is a recently identified family of cAMP and cGMP hydrolyzing enzymes. Thus far, a single splice variant designated as PDE11A1 has been reported. In this study, we identify and characterize two additional splice variants of PDE11A, PDE11A2 and PDE11A3. The full-length cDNAs are 2,141 bp for PDE11A2 and 2205 bp for PDE11A3. The ORF of PDE11A2 predicts a protein of 576 aa with a molecular mass of 65.8 kDa. The ORF of PDE11A3 predicts a protein of 684 aa with a molecular mass of 78.1 kDa. Comparison of the PDE11A2 sequence with that of PDE11A1 indicates an additional 86 aa at the N terminus of PDE11A2. Part of this sequence extends the potential cGMP binding region (GAF domain) present in PDE11A1. Compared with PDE11A2, PDE11A3 has an additional 108 N-terminal amino acids. Sequence analysis of PDE11A3 indicates the presence of another GAF domain in this region. This diversification of regulatory sequences in the N-terminal region of PDE11A splice variants suggests the interesting possibility of differential regulation of these enzymes. Recombinant PDE11A2 and -A3 proteins expressed in the Baculovirus expression system have the ability to hydrolyze both cAMP and cGMP. The [K.sub.m] values for cAMP hydrolysis are 3.3 [micro]M and 5.7 [micro]M for PDE11A2 and PDE11A3, respectively. The [K.sub.m] values for cGMP hydrolysis are 3.7 [micro]M and 4.2 [micro]M for PDE11A2 and PDE11A3, respectively. Both PDEs showed a [V.sub.max] ratio for cAMP/cGMP of approximately 1.0. PDE11A2 is sensitive to dipyridamole, with an [IC.sub.50] of 1.8 [micro]M, and to zaprinast, with an [IC.sub.50] of 28 [micro]M. PDE11A3 demonstrated similar pattern of inhibitor sensitivity with [IC.sub.50] values of 0.82 and 5 [micro]M for dipyridamole and zaprinast, respectively.

The PDE inhibitor zaprinast enhances NO-mediated protection against vascular leakage in reperfused lungs. Am J Physiol Lung Cell Mol Physiol 279: L496-L502, 2000.--Disruption of endothelial barrier properties with development of noncardiogenic pulmonary edema is a major threat in lung ischemiareperfusion (I/R) injury that occurs under conditions of lung transplantation. Inhaled nitric oxide (NO) reduced vascular leakage in lung I/R models, but the efficacy of this agent may be limited. We coadministered NO and zaprinast, a cGMP-specific phosphodiesterase inhibitor, to further augment the NO-cGMP axis. Isolated, buffer-perfused rabbit lungs were exposed to 4.5 h of warm ischemia. Reperfusion provoked a transient elevation in pulmonary arterial pressure and a negligible rise in microvascular pressure followed by a massive increase in the capillary filtration coefficient and severe lung edema formation. Inhalation of 10 parts/million of NO or intravascular application of 100 [micro]M zaprinast on reperfusion both reduced pressor response and moderately attenuated vascular leakage. Combined administration of both agents induced no additional vasodilation at constant microvascular pressures, but additively protected against capillary leakage paralleled by a severalfold increase in perfusate cGMP levels. In conclusion, combining low-dose NO inhalation and phosphodiesterase inhibition may be suitable for the maintenance of graft function in lung transplantation by amplifying the beneficial effect of the NO-cGMP axis and avoiding toxic effects of high NO doses. phosphodiesterase; nitric oxide; lung transplantation; cyclic nucleotides; microvascular permeability

Muscarinic cholinergic signaling plays an essential role in the control of the normal airway functions and in the development of pulmonary pathologies including asthma. In this paper we demonstrate that the airways of mice deficient in a cAMP-specific phosphodiesterase (PDE4D) are no longer responsive to cholinergic stimulation. Airway hyperreactivity that follows exposure to antigen was also abolished in [PDE4D.sup.-/-] mice, despite an apparently normal lung inflammatory infiltration. The loss of cholinergic responsiveness was specific to the airway, not observed in the heart, and was associated with a loss of signaling through muscarinic receptors with an inability to decrease cAMP accumulation. These findings demonstrate that the PDE4D gene plays an essential role in cAMP homeostasis and cholinergic stimulation of the airway, and in the development of hyperreactivity. In view of the therapeutic potentials of PDE4 inhibitors, our findings provide the rationale for novel strategies that target a single PDE isoenzyme.

A member of the phosphodiesterase (PDE)7 family with high affinity and specificity for cAMP has been identified. Based on sequence homologies, we designate this PDE as PDE7B. The full-length cDNA of PDE7B is 2399 bp, and its ORF sequence predicts a protein of 446 amino acids with a molecular mass of 50.1 kDa. Comparison of the predicted protein sequences of PDE7A and PDE7B reveals an identity of 70% in the catalytic domain. Northern blotting indicates that the mRNA of PDE7B is 5.6 kb. It is most highly expressed in pancreas followed by brain, heart, thyroid, skeletal muscle, eye, ovary, submaxillary gland, epididymus, and liver. Recombinant PDE7B protein expressed in a Baculovirus expression system is specific for cAMP with a [K.sub.m] of 0.03 [micro]M. Within a series of common PDE inhibitors, it is most potently inhibited by 3-isobutyl-1-methylxanthine with an [IC.sub.50] of 2.1 [micro]M. It is also inhibited by papaverine, dipyridamole, and SCH51866 at higher doses. PDE7A and PDE7B exhibit the same general pattern of inhibitor specificity among the several drugs tested. However, differences in [IC.sub.50] for some of the drugs suggest that isozyme selective inhibitors can be developed.

Two cloned soybean calmodulin isoforms, SCaM-4 and SCaM-1, were almost similar in their activation of phosphodiesterase, but only SCaM-1 activated nicotinamide adenine dinucleotide kinase. This differential regulation was further investigated by examining the capacity of SCaM-1 and SCaM-4 to activate mammalian nitric oxide synthase and calmodulin (CaM). Results indicated that the isoforms show a differential ability to activate enzymes and that they can serve as selective, competitive antagonists of certain CaM target enzymes.

Safety Study of Clinical and Immune Effects of Phosphodiesterase 4 (PDE-4) Inhibitor in Cutaneous Lupus Patients Sponsored by New York University School of Medicine. Collaborator: Celgene Corporation. Discoid cutaneous lupus [...]

The structure and stability of a novel Cr(III)-phosphodiester complex prepared in aqueous solution were investigated. The complex, cis-(Cr(phen)2(OP(O)(OC6H5)2)(H2O))2+, was shown to be remarkably stable over a wide range of pHs and may serve as a model for the interaction of Cr(III) compounds with DNA and for the stable adducts formed in these reactions. Furthermore, the diphenyl phosphate is bound to the Cr(III) ion in an anti, unidentate manner.

The effect on binding and specificity of a positively charged backbone with hydrophobicity using a polycationic nucleotide link with methylisothiouronium salts or methylated thioureas or DNmt was examined. These resulted to the formation of pentameric thymidyl deoxyribonucleic methylthiourea, in which the phosphodiester linkages of DNA were replaced by methylated thioureas. Moreover, these suggested that that the link between negatively charged DNA or RNA with positively charged DNmt contribute to the stability of heteroduplex and -triplex structures formed between them.

A study was conducted on the uncatalyzed rates of hydrolysis of unactivated monoesters and diesters of phosphoric acid as well as the proficiencies of phosphodiesterases and phosphatases as catalysts. The results indicate that the transition state affinities of phosphoric ester hydrolases approach the extremely low value of 5 x 10 (super -24) M. Moreover, phosphodiesters are very appropriate for the storage of genetic information.

The first metalloreceptor with a neutral bis(acylaminoimidazoline) binding site, which recognizes anionic and neural phosphodiesters through luminescent signal transduction, is reported. The metalloreceptor formed a complex with neutral phosphodiesters, a reaction significantly dependent on guest acidity and solvents. Its neutral bis(acylaminoimidazoline) binding site was found to act as both hydrogen-bond donors and acceptors. The luminescent response of the metalloreceptor to the complex formation demonstrated the optical sensing of photodiesters.

The tricyclo-DNA was examined to determine the effect of structural preorganization of DNA single strands on their pairing properties. Results indicated that tricyclo-DNA is a phosphodiester-backbone based DNA analog exhibiting strong complementary base-pairing properties. Furthermore, tricyclo DNA can be efficiently synthesized by using phosphoramidite chemistry.

Research is presented investigating the characteristics of phosphodiester oligodeoxynucleotides (ODNs) incorporating 4'alpha-C-aminomethyl, -N-(2-aminoethyl)carbamoylthymidines, -propyl and -ethyl as potential antisense molecules.

Research was conducted to examine the ability of natural polyamines, putrescine, spermidine and spermine and a series of their structural analogues to stabilize RNA-DNA hybrids using melting temperature measurements, circular dichroism spectroscopy and the ethidium bromide displace assay. Results suggest that the hexamine and pentamine analogues of spermine may be useful in improving the efficacy of therapeutic oligodeoxyribonucleotides.

The alpha6beta4 integrin initiates carcinoma invasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway. A study was conducted to show that alpha6beta4 causes chemotactic migration, a major component of invasion, by employing MDA-MB-435 breast carcinoma cells. However, it has no effect on haptotaxis. The integrin initiates the chemotactic migration of carcinoma cells by its capacity to affect major signaling events that are associated with carcinoma invasion.

Research was conducted to examine the hypothesis that persistent or increased phosphodiesterases (PDE5) may have contributed to the persistently elevated pulmonary vascular resistance in persistent pulmonary hypertension of the newborn (PPHN). The physiological effects of the PDE5 antagonists zaprinast and dipyridamole in fetal lambs with intrauterine pulmonary hypertension and in age-matched control lambs. Results indicate that PDE5 activity was increased in experimental PPHN, possibly by posttranslational phosphorylation.