Michael Sullivan

The Human Cyclic AMP-specific Phosphodiesterase PDE-46 (HSPDE4A4B) Expressed in Transfected COS7 Cells Occurs as Both Particulate and Cytosolic Species That Exhibit Distinct Kinetics of Inhibition by the Antidepressant Rolipram

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed ∼96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a ∼125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmax of particulate PDE-46 was ∼56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an ∼11-fold higher Vmax relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0.195 μM) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 μM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki = 1.6 μM) but as a partial competitive inhibitor of the particulate enzyme (Ki = 0.037 μM; Ki′ = 2.3 μM). Particulate PDE-46 thus showed a ∼60-fold higher affinity for rolipram than cytosolic PDE-46.

Molecular cloninc and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1)

Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE. Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference. The h6.1 cDNA was extended at the 5-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids. The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3-untranslated region, with the previously reported human PDE-IVA cDNA [Livi G. P., Kmetz P., Mchale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J. and Balcarek J. M. (1990) Mol. Cell Biol. 10, 2678-2686]. The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals. The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain. The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells. Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S. cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype. Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour. Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression, intracellular distribution and basis for lack of catalytic activity of the PDE4A7 isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene

PDE4A7 is an isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene that fails to hydrolyse cAMP and whose transcripts are widely expressed. Removal of either the N- or C-terminal unique portions of PDE4A7 did not reconstitute catalytic activity, showing that they did not exert a chronic inhibitory effect. A chimera (Hyb2), formed by swapping the unique N-terminal portion of PDE4A7 with that of the active PDE4A4C form, was not catalytically active. However, one formed (Hyb1) by swapping the unique C-terminal portion of PDE4A7 with that common to all active PDE4 isoforms was catalytically active. Compared with the active PDE4A4B isoform, Hyb1 exhibited a similar K(m) value for cAMP and IC50 value for rolipram inhibition, but was less sensitive to inhibition by Ro-20-1724 and denbufylline, and considerably more sensitive to thermal denaturation. The unique C-terminal region of PDE4A7 was unable to support an active catalytic unit, whereas its unique N-terminal region can. The N-terminal portion of the PDE4 catalytic unit is essential for catalytic activity and can be supplied by either highly conserved sequence found in active PDE4 isoforms from all four PDE4 subfamilies or the unique N-terminal portion of PDE4A7. A discrete portion of the conserved C-terminal region in active PDE4A isoforms underpins their aberrant migration on SDS/PAGE. Unlike active PDE4A isoforms, PDE4A7 is exclusively localized to the P1 particulate fraction in cells. A region located within the C-terminal portion of active PDE4 isoforms prevents such exclusive targeting. Three functional regions in PDE4A isoforms are identified, which influence catalytic activity, subcellular targeting and conformational status.

Genomic Organisation of the Human Cyclic AMP-Specific Phosphodiesterase PDE4C Gene and Its Chromosomal Localisation to 19p13.1, Between RAB3A and JUND¶

PDE4C is one of four mammalian genes that encode multiple PDE4 cyclic AMP-specific phosphodiesterase isoforms that are inhibited by rolipram. Fluorescent in situ hybridisation localised PDE4C to the p13.1 region of human chromosome 19. Overlapping cosmid clones spanning the human PDE4C gene were identified and characterised. Analysis of this locus indicated that the PDE4C gene spans at least 38 kb, consists of at least 18 exons, and contains the marker D19S212 within an intron. Comparison of published human PDE4C cDNA sequences with those of the genomic DNA identified four alternatively spliced exons and the possibility that the PDE4C locus contains at least three alternative promoters. PDE4C-containing cosmids also contained the genes for the growth regulatory transcription factor, JUND, and the mini guanine nucleotide regulatory protein, RAB3A. The RAB3A gene was shown to consist of 5 exons spanning 7.9 kb, while the JUND gene was found to contain no introns. Analysis of cosmids containing PDE4C, JUND, and RAB3A showed that 27 kb separate JUND and PDE4C, while only 3.7 kb separate PDE4C and RAB3A. The three genes share the same orientation of transcription and are arranged in the order cen- 5- JUND-PDE4C-RAB3A-3-tel.

Book review: The Democratic Plan: Analysis and Diagnosis

stepping stones provided in Dávila’s book. Switching to a far more pessimistic tone, the third section of the book discusses the plan for building an aerial cable-car line in Soacha (Cazucable), a relatively large autonomous municipality adjacent to Bogotá. The five chapters in this section discuss, in a rather overlapping manner, the political, administrative and financial pitfalls faced by the project. The story of the Cazucable is one of lack of integration of city administrations despite the evident functional integration of Bogotá and Soacha, coupled with geological conditions that call for disaster. Evidently, sound planning, institutional capacity and a broader view of transportrelated interventions are called for in order to meet the high expectations of the residents of the affected areas. The fourth section is devoted to the discussion of lessons from two plans to build similar projects in Cali and La Paz, and two running aerial cable-cars in Caracas and Rio de Janeiro. The chronic delays faced by these projects highlight the key role of continuous political support in ensuring the implementation of plans. The chapters on the lessons from Caracas and Rio de Janeiro do not offer comparable impact estimates, but they do suggest that the impacts may not be sizable given the overestimation of the demand for this particular mode of transportation. The last section of the book concludes by discussing the effectiveness of interventions based on three inter-related elements: increase in mobility, reduction in poverty and urban integration of the impacted areas with the rest of the city. The first criterion is fairly quantifiable and comparable across time and cities, and would reflect the effect of offering faster, safer but still affordable alternatives to existing means of transportation. On the other hand, reductions in poverty caused by transport-related interventions and complementary projects are far more difficult to pinpoint. As it becomes clear after reading Dávila’s book, so far we do not have solid comparable empirical evidence on the magnitude of the reductions in poverty and inequality that can be attributed to different interventions, nor do we have a strong enough theoretical basis for understanding the channels through which these effects operate. Would, for instance, a combined strategy of localised formal jobs creation and increased transportation access be a better alternative for reducing poverty? Finally, the last element is less tangible because of its symbolic nature, but may be far more crucial with regards to the sustained impact of similar interventions. The change in attitudes following the interventions may be a key element in the narrowing of segregation and exclusion in the long term, even if there is not an apparent improvement in social indicators in the short term. As the Medellı́n example shows, interventions centred on transportation infrastructure provision can be a means to this end, but whether they gain enough momentum or fade out is a political matter.