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The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay. The interaction was confirmed in biochemical pull-down analyses. The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52. XAP2 also did not interact with other PDE4A isoforms or typical isoforms from the three other PDE4 subfamilies. Functionally, XAP2 reversibly inhibited the enzymatic activity of PDE4A5, increased the sensitivity of PDE4A5 to inhibition by the prototypical PDE4 inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and attenuated the ability of cAMP-dependent protein kinase to phosphorylate PDE4A5 in intact cells. XAP2 maximally inhibited PDE4A5 by approximately 60%, with an IC50 of 120 nm, and reduced the IC50 for rolipram from 390 nm to 70-90 nm. Co-expression of XAP2 and PDE4A5 in COS7 cells showed that they could be co-immunoprecipitated and also reduced both the enzymatic activity of PDE4A5 and its IC50 for rolipram. Native XAP2 and PDE4A5 could be co-immunoprecipitated from the brain. The isolated COOH-terminal half of XAP2 (amino acids 170-330), containing its tetratricopeptide repeat domain, but not the isolated NH2-terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC50 for rolipram. Mutation of Arg271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in two-hybrid assays and to inhibit PDE4A5 activity. Either the deletion of a specific portion of the unique amino-terminal region or specific mutations in the regulatory UCR2 domain of PDE4A5 attenuated its ability be inhibited by XAP2. We suggest that XAP2 functionally interacts with PDE4A5 in cells.

The PDE4 cyclic AMP-specific phosphodiesterase family comprises a large number of different isoforms encoded by four distinct genes, with additional complexity arising through alternate mRNA splicing. This generates a number of distinct PDE4 isoforms with unique N-terminal regions. The range of such splice variants emanating from the four PDE4 genes appears to be highly conserved across species. One key role for such regions appears to be their potential to target isoforms to specific intracellular sites. Evidence for such a targeting role for these N-terminal regions can be gleaned by a variety of techniques. These include subcellular fractionation, confocal microscopy, binding assays to show association with proteins having src homology 3 (SH3) domains, and generation of chimeric constructs of these N-terminal regions with proteins that are normally expressed in the cytosol.

Variant Creutzfeldt-Jakob disease (vCJD) was first identified in 1996 in the UK, and results from human exposure to the bovine spongiform encephalopathy (BSE) agent. vCJD has subsequently been identified in 10 additional countries, and numbers continue to increase in the UK. Unlike other human prion diseases, infectivity and the disease-associated form of the prion protein are readily detected in lymphoid tissues in vCJD. In experimental BSE infection in a sheep model, infectivity has been transmitted by blood transfusion from asymptomatic infected animals to normal recipient animals, indicating that infectivity is present in blood during the incubation period. Recently, two cases of apparent iatrogenic vCJD infection by blood transfusion from asymptomatic donors who subsequently died from vCJD have been reported from the UK. The first case resulted in clinical illness identical to other cases of vCJD, while the second case was an asymptomatic infection detected at autopsy. Sensitive means of detection of disease-associated prion protein in the blood are required in order to be employed for screening purposes, both individually at the time of blood donation, and to help ascertain future numbers of vCJD cases in the UK and beyond.

Variant Creutzfeldt-Jakob disease (vCJD) is a novel human prion disease that results from exposure to the bovine spongiform encephalopathy (BSE) agent, probably by the oral route. The pathological features of vCJD are unique, with extensive involvement of lymphoid tissues in addition to the central nervous system. This article reviews the histopathology and biochemistry of vCJD, emphasising diagnostic features and indicating several areas of active research. The widespread distribution of infectivity in lymphoid tissues in vCJD has lead to concerns over the possibility of iatrogenic disease transmission by contaminate surgical instruments, or by blood transfusion. vCJD has so far only occurred in individuals within a genetic subset defined by the natural polymorphism at codon 129 in the prion protein gene. It remains uncertain if this disease will occur in other genetic subgroups within the population. Continuing surveillance of vCJD in the UK and other countries in which BSE has been identified will be necessary for future estimations of disease numbers worldwide.

The challenge of 3T3-F442A fibroblasts with growth hormone led to both a decrease in the mobility on SDS/PAGE and activation of the PDE4A cyclic AMP-specific phosphodiesterase isoform PDE4A5. Activation was mediated by a JAK-2-dependent pathway coupled to the activation of phosphatidylinositol 3-kinase and p70S6 kinase. Activation was not dependent on the ability of growth hormone to stimulate ERK2 or protein kinase C or any effect on transcription. Blockade of activation of murine PDE4A5 ablated the ability of growth hormone to decrease intracellular cAMP levels. Antisense depletion of murine PDE4A5 mimicked the ability of rolipram to enhance the growth hormone-stimulated differentiation of 3T3-F442A cells to adipocytes. It is suggested that activation of PDE4A5 by growth hormone serves as a brake on the differentiation processes.