Cloning and characterization of two splice variants of human phosphodiesterase 11A.

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(m) values for cAMP hydrolysis are 3.3 microM and 5.7 microM for PDE11A2 and PDE11A3, respectively. The K(m) values for cGMP hydrolysis are 3.7 microM and 4.2 microM for PDE11A2 and PDE11A3, respectively. Both PDEs showed a V(max) ratio for cAMP/cGMP of approximately 1.0. PDE11A2 is sensitive to dipyridamole, with an IC(50) of 1.8 microM, and to zaprinast, with an IC(50) of 28 microM. PDE11A3 demonstrated similar pattern of inhibitor sensitivity with IC(50) values of 0.82 and 5 microM for dipyridamole and zaprinast, respectively.

Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A.

We report here the cloning, expression, and characterization of human PDE11A1, a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits

Cloning and characterization of PDE7B, a cAMP-specific phosphodiesterase.

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(m) of 0.03 microM. Within a series of common PDE inhibitors, it is most potently inhibited by 3-isobutyl-1-methylxanthine with an IC(50) of 2.1 microM. 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(50) for some of the drugs suggest that isozyme selective inhibitors can be developed.

Is glass visible on plain radiographs? A cadaver study.

Eighteen randomly selected pieces of nonleaded glass from a collection of 30 pieces from broken bottles of four known color types found on the streets of Houston were sorted into four sets with one control. The purpose of this study was to determine if regular glass is visible on plain radiographs and whether color, location, or volume of these fragments had any effect on the detection of nonleaded glass. These sets were then placed into a fresh-frozen cadaver foot and ankle with a history of insulin-dependent diabetes and peripheral vascular disease. This foot was radiographed utilizing four standard foot projections. Five examiners read these five sets of radiographs twice and recorded the number seen. Overall sensitivity for all of the examiners was 90% with an overall false-positive rate of 10%. Intraobserver and interobserver Pearson's correlation coefficients showed that there was reliability between the first and second readings and between observers. The authors concluded that regular nonleaded glass is radiographically visible and that factors such as color and location of the glass have no effect on its detection, while a volume of less than 15 mm3 may have an effect on the detection of glass.

Inhibition of in vitro proliferation of chronic myelogenous leukemia progenitor cells by c-myb antisense oligodeoxynucleotides.

Normal hematopoietic progenitors and acute myelogenous leukemia cells show a differential requirement for the encoded product of c-myb proto-oncogene for proliferation. To determine whether c-myb is also differentially required for the proliferation of hematopoietic progenitors of chronic myelogenous leukemia (CML), mononuclear cells derived from both chronic phase and blast crisis were exposed to c-myb antisense oligodeoxynucleotides and assayed for colony-forming ability. Exposure of CML-BC cells from 12 patients to c-myb antisense oligodeoxynucleotides resulted in significant (p<001) inhibition of leukemia colony formation (average inhibition 63%) and was accompanied by down-regulation of c-myb expression. Colonies derived from CML chronic phase progenitors were virtually unaffected in 10 cases, but down-regulation of c-myb expression was not detected. However, in studies conducted with CD34+ leukemia cells, a subset highly enriched for hematopoietic progenitors, colony formation was inhibited at both disease stages, whereas CFU-GM colony formation derived from normal CD34+ cells was not affected by exposure to c-myb antisense oligodeoxynucleotides. These data suggest that CML chronic phase and blast crisis progenitors are both sensitive to the inhibitory effects of c-myb antisense oligomers, and that the lack of inhibition in partially purified CML-chronic phase progenitors is probably due to inefficient penetration of oligodeoxynucleotides into the clonogenic cells. The preferential effect of c-myb antisense oligodeoxynucleotides on colonies arising from the compartment that includes CML-CD34+ progenitors likely reflects the expansion of a cell population with high proliferative potential and elevated c-myb mRNA levels.

Cutaneous foot depigmentation following an intra-articular steroid injection.

Local skin depigmentation is a potentially troublesome side effect of local steroid injection. Although many authors believe that it is a rare occurrence, this rarity may be a result of the low number of cases that are actually reported. The authors are of the opinion that the practitioner should take the time to explain depigmentation as a possible side effect of local steroid injection to those patients whose skin color places them at cosmetic risk.