Phosphorylation of PDE4A5 by MAPKAPK2 attenuates fibrin degradation via p75 signalling.

Phosphodiesterases (PDEs) shape local cAMP gradients to underpin the specificity of receptor function. Key to this process is the highly defined nature of the intra-cellular location of PDEs in the cell. PDE4A5 is a PDE isoform that specifically degrades cAMP and is known to associate with the p75 neurotrophin receptor (p75NTR) where it modulates cAMP signalling cascades that regulate extracellular matrix remodelling in the lungs. Here we map and validate novel protein-protein interaction sites that are important for formation of the PDE4A5-p75NTR complex and show, for the first time, that phosphorylation of PDE4A5 by MAPKAPK2 enhances PDE4A5 interaction with p75NTR and that this, in turn, serves to attenuate fibrin degradation.

FBXW7 regulates DISC1 stability via the ubiquitin-proteosome system.

Disrupted in schizophrenia 1 (DISC1) is a multi-functional scaffolding protein that has been associated with neuropsychiatric disease. The role of DISC1 is to assemble protein complexes that promote neural development and signaling, hence tight control of the concentration of cellular DISC1 in neurons is vital to brain function. Using structural and biochemical techniques, we show for we believe the first time that not only is DISC1 turnover elicited by the ubiquitin proteasome system (UPS) but that it is orchestrated by the F-Box protein, FBXW7. We present the structure of FBXW7 bound to the DISC1 phosphodegron motif and exploit this information to prove that disruption of the FBXW7-DISC1 complex results in a stabilization of DISC1. This action can counteract DISC1 deficiencies observed in neural progenitor cells derived from induced pluripotent stem cells from schizophrenia patients with a DISC1 frameshift mutation. Thus manipulation of DISC1 levels via the UPS may provide a novel method to explore DISC1 function.

Human phosphodiesterase 4D7 (PDE4D7) expression is increased in TMPRSS2-ERG-positive primary prostate cancer and independently adds to a reduced risk of post-surgical disease progression.

BACKGROUND: There is an acute need to uncover biomarkers that reflect the molecular pathologies, underpinning prostate cancer progression and poor patient outcome. We have previously demonstrated that in prostate cancer cell lines PDE4D7 is downregulated in advanced cases of the disease. To investigate further the prognostic power of PDE4D7 expression during prostate cancer progression and assess how downregulation of this PDE isoform may affect disease outcome, we have examined PDE4D7 expression in physiologically relevant primary human samples. METHODS: About 1405 patient samples across 8 publically available qPCR, Affymetrix Exon 1.0 ST arrays and RNA sequencing data sets were screened for PDE4D7 expression. The TMPRSS2-ERG gene rearrangement status of patient samples was determined by transformation of the exon array and RNA seq expression data to robust z-scores followed by the application of a threshold>3 to define a positive TMPRSS2-ERG gene fusion event in a tumour sample. RESULTS: We demonstrate that PDE4D7 expression positively correlates with primary tumour development. We also show a positive association with the highly prostate cancer-specific gene rearrangement between TMPRSS2 and the ETS transcription factor family member ERG. In addition, we find that in primary TMPRSS2-ERG-positive tumours PDE4D7 expression is significantly positively correlated with low-grade disease and a reduced likelihood of progression after primary treatment. Conversely, PDE4D7 transcript levels become significantly decreased in castration resistant prostate cancer (CRPC). CONCLUSIONS: We further characterise and add physiological relevance to PDE4D7 as a novel marker that is associated with the development and progression of prostate tumours. We propose that the assessment of PDE4D7 levels may provide a novel, independent predictor of post-surgical disease progression.

Cholinergic deregulation in traumatic brain injury could be a pathophysiology-related biphasic epiphenomenon.

No abstract available.

Phosphodiesterase 4 interacts with the 5-HT4(b) receptor to regulate cAMP signaling.

Phosphodiesterase (PDE) 3 and PDE4, which degrade cyclic adenosine monophosphate (cAMP), are important regulators of 5-hydroxytryptamine (5-HT) 4 receptor signaling in cardiac tissue. Therefore, we investigated whether they interact with the 5-HT4(b) receptor, and whether A-kinase anchoring proteins (AKAPs), scaffolding proteins that bind to the regulatory subunit of protein kinase A (PKA) and contribute to the spacial-temporal control of cAMP signaling, are involved in the regulation of 5-HT4(b) receptor signaling. By measuring PKA activity in the absence and presence of PDE3 and PDE4 inhibitiors, we found that constitutive signaling of the overexpressed HA-tagged 5-HT4(b) receptor in HEK293 cells is regulated predominantly by PDE4, with a secondary role for PDE3 that is unmasked in the presence of PDE4 inhibition. Overexpressed PDE4D3 and PDE3A1, and to a smaller extent PDE4D5 co-immunoprecipitate constitutively with the 5-HT4(b) receptor. PDE activity measurements in immunoprecipitates of the 5-HT4(b) receptor confirm the association of PDE4D3 with the receptor and provide evidence that the activity of this PDE may be increased upon receptor stimulation with 5-HT. A possible involvement of AKAPs in 5-HT4(b) receptor signaling was uncovered in experiments using the St-Ht31 inhibitor peptide, which disrupts the interaction of AKAPs with PKA. However, St-Ht31 did not influence 5-HT4(b) receptor-stimulated PKA activity, and endogenous AKAP79 and gravin were not found in immunoprecipitates of the 5-HT4(b) receptor. In conclusion, we found that both PDE3A1 and PDE4D3 are integrated into complexes that contain the 5-HT4(b) receptor and may thereby regulate 5-HT4(b) receptor-mediated signaling.

The cAMP phosphodiesterase-4D7 (PDE4D7) is downregulated in androgen-independent prostate cancer cells and mediates proliferation by compartmentalising cAMP at the plasma membrane of VCaP prostate cancer cells.

BACKGROUND: Isoforms of the PDE4 family of cAMP-specific phosphodiesterases (PDEs) are expressed in a cell type-dependent manner and contribute to underpinning the paradigm of intracellular cAMP signal compartmentalisation. Here we identify the differential regulation of the PDE4D7 isoform during prostate cancer progression and uncover a role in controlling prostate cancer cell proliferation. METHODS: PDE4 transcripts from 19 prostate cancer cell lines and xenografts were quantified by qPCR. PDE4D7 expression was further investigated because of its significant downregulation between androgen-sensitive (AS) and androgen-insensitive (AI) samples. Western blot analysis, PDE activity assay, immunofluorescent staining and cAMP responsive FRET assays were used to investigate the sub-plasma membrane localisation of a population of PDE4D7 in VCaP (AS) and PC3 (AI) cell lines. Disruption of this localisation pattern using dominant-negative protein expression and siRNA knockdown showed that PDE4D7 acts in opposition to proliferative signalling as assessed by electrical impedance-based proliferation assays. RESULTS: Here we identify the differential regulation of the PDE4D7 isoform during prostate cancer progression. PDE4D7 is highly expressed in AS cells and starkly downregulated in AI samples. The significance of this downregulation is underscored by our finding that PDE4D7 contributes a major fraction of cAMP degrading PDE activity tethered at the plasma membrane and that displacement of PDE4D7 from this compartment leads to an increase in the proliferation of prostate cancer cells. PDE4D7 mRNA expression is not, however, directly regulated by the androgen receptor signalling axis despite an overlapping genomic structure with the androgen responsive gene PART1. PDE4D7, which locates to the plasma membrane, acts to supress aberrant non-steroidal growth signals within the prostate or AS metastasis. CONCLUSIONS: PDE4D7 expression is significantly downregulated between AS and AI cell phenotypes. This change in expression potentially provides a novel androgen-independent biomarker and manipulation of its activity or its expression may provide therapeutic possibilities and insights into contributory aspects of the complex molecular pathology of prostate cancer.

Estimating reassortment rates in co-circulating Eurasian swine influenza viruses.

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2-3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

Long-term experience with everolimus in kidney transplantation in the United States.

BACKGROUND: Limited long-term data exist on US kidney transplant patients who have received everolimus at time of transplantation. METHODS: Using data from the United Network for Organ Sharing/Organ Procurement Transplant Network database, we described patient characteristics and outcomes among adult patients who received a kidney transplant between 1998 and 2007 and received everolimus maintenance immunosuppression (n = 392) at time of discharge. Outcomes included acute rejection, new-onset diabetes posttransplant, primary graft failure, and serum creatinine. We included single-organ, first-time transplants between 1998 and 2007 as a reference group. RESULTS: Primary graft survival at 3 and 5 years posttransplantation was 87.2% ± 2.1% (95% confidence interval [CI]: 82.5%-90.7%) and 77.4% ± 3.0% (95% CI: 70.8%-82.7%), respectively, in the everolimus-treated group. Improved graft survival with everolimus seemed to be more pronounced in recipients of deceased donor transplants despite the fact that everolimus-treated patients quantitatively had a higher rate of acute rejection at 3 years posttransplant versus the reference group. CONCLUSION: Although the incidence of acute rejection was slightly higher in the everolimus-treated patients, graft survival at 3 and 5 years posttransplantation favored everolimus, with the effect being particularly notable in the recipients who received deceased donor renal transplants.

The emerging role of HSP20 as a multifunctional protective agent.

The small heat shock proteins (sHSPs) are a highly conserved family of molecular chaperones that are ubiquitously expressed throughout nature. They are transiently upregulated in many tissue types following stressful stimuli. Recently, one member of the sHSP family, HSP20 (HspB6), has been shown to be highly effective as a protective mediator against a number of debilitating pathological conditions, including cardiac hypertrophy and Alzheimer's disease. Hsp20 is also an important modulator of vital physiological processes, such as smooth muscle relaxation and cardiac contractility. This review focuses on the molecular mechanisms employed by HSP20 that allow it to act as an innate protector in the context of cardiovascular and neurological diseases. Emerging evidence for a possible role as an anti-cancer agent is also presented.

Disruption of the cyclic AMP phosphodiesterase-4 (PDE4)-HSP20 complex attenuates the ß-agonist induced hypertrophic response in cardiac myocytes.

The small heat shock protein HSP20 is known to be cardioprotective during times of stress and the mechanism underlying its protective abilities depends on its phosphorylation on Ser16 by PKA (protein kinase A). Although the external stimuli that trigger Ser16 phosphorylation have been well studied, the events that modulate spatial and temporal control of this modification remain to be clarified. Here, we report that inhibition of cAMP phosphodiesterase-4 (PDE4) induces the phosphorylation of HSP20 in resting cardiac myocytes and augments its phosphorylation by PKA following ß-adrenergic stimulation. Moreover, using peptide array technology, in vitro binding studies, co-immunoprecipitation techniques and immunocytochemistry, we show that HSP20 binds directly to PDE4 within a region of the conserved catalytic domain. We also show that FRET-based, genetically-encoded cAMP reporters anchored to HSP20 exhibit a larger response to PDE4 inhibition compared to free cytosolic cAMP reporters, suggesting that the interaction with PDE4 is crucial in modulating the highly localised pool of cAMP to which HSP20 is exposed. Using information gleaned from peptide array analyses, we developed a cell-permeable peptide that serves to inhibit the interaction of PDE4 with HSP20. Disruption of the HSP20-PDE4 complex, using this peptide, suffices to induce phosphorylation of HSP20 by PKA and to protect against the hypertrophic response measured in neonatal cardiac myocytes following chronic ß-adrenergic stimulation.

ß-Arrestin 1 inhibits the GTPase-activating protein function of ARHGAP21, promoting activation of RhoA following angiotensin II type 1A receptor stimulation.

Activation of the small GTPase RhoA following angiotensin II stimulation is known to result in actin reorganization and stress fiber formation. Full activation of RhoA, by angiotensin II, depends on the scaffolding protein ß-arrestin 1, although the mechanism behind its involvement remains elusive. Here we uncover a novel partner and function for ß-arrestin 1, namely, in binding to ARHGAP21 (also known as ARHGAP10), a known effector of RhoA activity, whose GTPase-activating protein (GAP) function it inhibits. Using yeast two-hybrid screening, a peptide array, in vitro binding studies, truncation analyses, and coimmunoprecipitation techniques, we show that ß-arrestin 1 binds directly to ARHGAP21 in a region that transects the RhoA effector GAP domain. Moreover, we show that the level of a complex containing ß-arrestin 1 and ARHGAP21 is dynamically increased following angiotensin stimulation and that the kinetics of this interaction modulates the temporal activation of RhoA. Using information gleaned from a peptide array, we developed a cell-permeant peptide that serves to inhibit the interaction of these proteins. Using this peptide, we demonstrate that disruption of the ß-arrestin 1/ARHGAP21 complex results in a more active ARHGAP21, leading to less-efficient signaling via the angiotensin II type 1A receptor and, thereby, attenuation of stimulated stress fiber formation.

Enteric-coated mycophenolate sodium versus mycophenolate mofetil maintenance immunosuppression: outcomes analysis of the United Network for Organ Sharing/Organ Procurement and Transplantation Network database.

BACKGROUND: A large, retrospective database analysis was conducted to evaluate the long-term outcomes of patients who received enteric-coated mycophenolate sodium (EC-MPS) versus mycophenolate mofetil (MMF) maintenance immunosuppression at the time of discharge. METHODS: All primary kidney transplant patients who received either EC-MPS or MMF at time of discharge in the United Network for Organ Sharing/Organ Procurement and Transplantation Network database from 2004 to 2007 were included. Patients were excluded if they had received a previous kidney transplant, multiple organs, or combination therapy with everolimus at the time of discharge. Outcomes included graft failure, death-censored graft failure, and death with functioning graft, biopsy-proven acute rejection (BPAR), new-onset diabetes mellitus, and renal function. The propensity score method was used to adjust for nonrandomized treatment selection. A total of 48,458 patients were included in the analysis. RESULTS: At time of discharge, 10.4% of patients received EC-MPS (n=5057) and 89.6% received MMF (n=43,401). Propensity score-adjusted regression analysis showed that patients who received EC-MPS were at increased risk of BPAR (hazards ratio, 1.167; 95% confidence interval, 1.056-1.129; P=0.002). CONCLUSIONS: The adjusted BPAR rate difference at 3 years posttransplantation was less than 2% (13.6% vs. 11.7%); statistically significant because of the large number of patients included in the analysis, but a difference that may not be clinically meaningful. No differences in graft survival, new-onset diabetes mellitus, or renal function were observed between the treatment groups.

Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis.

BACKGROUND AND PURPOSE: Apremilast is an orally administered phosphodiesterase-4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro-inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis. EXPERIMENTAL APPROACH: Apremilast was tested in vitro against endotoxin- and superantigen-stimulated PBMC, bacterial peptide and zymosan-stimulated polymorphonuclear cells, immunonoglobulin and cytokine-stimulated NK cells, and ultraviolet B light-activated keratinocytes. Apremilast was orally administered to beige-severe combined immunodeficient mice, xenotransplanted with normal human skin and triggered with human psoriatic NK cells. Epidermal skin thickness, proliferation index and inflammation markers were analysed. KEY RESULTS: Apremilast inhibited PBMC production of the chemokines CXCL9 and CXCL10, cytokines interferon-gamma and tumour necrosis factor (TNF)-alpha, and interleukins (IL)-2, IL-12 and IL-23. Production of TNF-alpha by NK cells and keratinocytes was also inhibited. In vivo, apremilast significantly reduced epidermal thickness and proliferation, decreased the general histopathological appearance of psoriasiform features and reduced expression of TNF-alpha, human leukocyte antigen-DR and intercellular adhesion molecule-1 in the lesioned skin. CONCLUSIONS AND IMPLICATIONS: Apremilast displayed a broad pattern of anti-inflammatory activity in a variety of cell types and decreased the incidence and severity of a psoriasiform response in vivo. Inhibition of TNF-alpha, IL-12 and IL-23 production, as well as NK and keratinocyte responses by this phosphodiesterase-4 inhibitor suggests a novel approach to the treatment of psoriasis.

Defining the risks for cytomegalovirus infection and disease after solid organ transplantation.

Cytomegalovirus continues to be one of the most clinically significant infections after solid organ transplantation. Classic definitions of patients at high risk for infection and tissue-invasive disease are focused on recipient-donor serostatus, type of organ transplanted, and overall level of immunosuppression. However, recent trends in clinical practice call for a reevaluation of cytomegalovirus infection risks after solid organ transplantation. Indeed, whereas early-onset cytomegalovirus infection is usually controlled by antiviral prophylaxis with ganciclovir and derivatives, delayed- and late-onset cytomegalovirus infection can develop after the completion of a course of preventive therapy. In addition, indirect effects of cytomegalovirus infection may occur as a result of persistent low-level viremia. Suboptimal dosing of antiviral drugs due to specific drug toxicities may result in the development of ganciclovir-resistant cytomegalovirus disease. The relationship between organ allograft rejection and cytomegalovirus infection and disease has been recognized for some time. Transplantation of increasing numbers of extended-criteria donor organs increases the risk of delayed graft function and acute rejection, prompting the use of more intensive immunosuppression. In addition, the trend to spare long-term exposure to calcineurin inhibitors has contributed to a resurgence in the use of polyclonal T-cell induction immunosuppressive agents, which may reduce host anticytomegalovirus immunity. We discuss the current trends in solid organ transplantation that provide a foundation for defining risks for cytomegalovirus infection and disease, including identification of patients who would benefit from more aggressive cytomegalovirus monitoring and prevention strategies.

The use of drotrecogin alfa (activated) in solid organ transplant patients: a case series.

BACKGROUND: Drotrecogin alfa (activated) (DAA), a recombinant human activated protein C, is indicated for the reduction of mortality in patients with severe sepsis who have a high risk of death. In the initial trial, DAA demonstrated a significant reduction in mortality at 28 days for patients treated with DAA in comparison with standard supportive treatment (placebo). However, solid organ transplant recipients were excluded from the study. Transplant recipients are at an increased risk for sepsis and there is minimal literature describing the safety and efficacy of DAA in the transplant population. METHODS: Thirteen solid organ transplant recipients who received DAA between November 2001 and January 2004 were included in this case series. Patients were prospectively identified and data collection occurred concurrently and by retrospective chart review. All patients met the DAA use criteria based on the institutional standard protocol. RESULTS: We report the outcomes of the 13 adult transplant patients who received a total of 14 courses of DAA for severe sepsis. At the time of DAA initiation, all patients required mechanical ventilation, 86% necessitated vasopressor support, and had a median of 3 dysfunctional organs. The median Acute Physiology and Chronic Health Evaluation (APACHE) II score at initiation was 30. Overall, hemodynamic stability and APACHE II score improved at the end of DAA infusion. Causes of early discontinuation were bleeding (57%), scheduled procedure (14%), increased international normalized ratio (14%), and death (14%). In-hospital, 28-day, and 1-year mortality was 69%, 62%, and 83%, respectively. CONCLUSION: DAA appears to be safe with appropriate monitoring. However, transplant recipients had a higher incidence of bleeding events leading to early discontinuation of DAA. Efficacy is difficult to assess without an appropriate control group for comparison.

No difference between smokers, former smokers, or nonsmokers in the operative outcomes of laparoscopic donor nephrectomies.

The laparoscopic donor nephrectomy has revolutionized the living donation process for kidney transplantation. Because this surgery is elective and altruistic and smoking has been associated with greater technical difficulty and increased risk for postoperative complications for other types of surgeries, the potential risk of smoking must be addressed with regard to surgical complications. We reviewed 221 laparoscopic kidney donors with known smoking status. Forty-two (19%) were smokers, 39 (18%) were former smokers, and 140 (63%) were nonsmokers. Important donor demographics were similar between groups. There was no difference between the 3 groups for mean operative time (4.5 h vs. 4.6 h vs. 4.4 h), median or mean length of stay (2 days for all groups), estimated blood loss (173+/-137 mL vs. 209+/-184 mL vs. 188+/-198 mL), narcotic use (0.57+/-0.48 mg/kg vs. 0.49+/-0.26 mg/kg vs. 0.53+/-0.36 mg/kg of total 4 morphine equivalents), or postoperative complications. Smoking status does not seem to impact perisurgical patient outcomes in patients undergoing laparoscopic nephrectomies.

The role of the PDE4D cAMP phosphodiesterase in the regulation of glucagon-like peptide-1 release.

BACKGROUND AND PURPOSE: Increases in intracellular cyclic AMP (cAMP) augment the release/secretion of glucagon-like peptide-1 (GLP-1). As cAMP is hydrolysed by cAMP phosphodiesterases (PDEs), we determined the role of PDEs and particularly PDE4 in regulating GLP-1 release. EXPERIMENTAL APPROACH: GLP-1 release, PDE expression and activity were investigated using rats and GLUTag cells, a GLP-1-releasing cell line. The effects of rolipram, a selective PDE4 inhibitor both in vivo and in vitro and stably overexpressed catalytically inactive PDE4D5 (D556A-PDE4D5) mutant in vitro on GLP-1 release were investigated. KEY RESULTS: Rolipram (1.5 mg x kg(-1) i.v.) increased plasma GLP-1 concentrations approximately twofold above controls in anaesthetized rats and enhanced glucose-induced GLP-1 release in GLUTag cells (EC(50) approximately 1.2 nmol x L(-1)). PDE4D mRNA transcript and protein were detected in GLUTag cells using RT-PCR with gene-specific primers and Western blotting with a specific PDE4D antibody respectively. Moreover, significant PDE activity was inhibited by rolipram in GLUTag cells. A GLUTag cell clone (C1) stably overexpressing the D556A-PDE4D5 mutant, exhibited elevated intracellular cAMP levels and increased basal and glucose-induced GLP-1 release compared with vector-transfected control cells. A role for intracellular cAMP/PKA in enhancing GLP-1 release in response to overexpression of D556A-PDE4D5 mutant was demonstrated by the finding that the PKA inhibitor H89 reduced both basal and glucose-induced GLP-1 release by 37% and 39%, respectively, from C1 GLUTag cells. CONCLUSIONS AND IMPLICATIONS: PDE4D may play an important role in regulating intracellular cAMP linked to the regulation of GLP-1 release.

Hematologic complications of anti-CMV therapy in solid organ transplant recipients.

Cytomegalovirus (CMV) infection complicates the post-operative course of patients receiving solid organ transplants. While ganciclovir has significantly reduced the direct effects of CMV infection, some patients cannot tolerate the optimal therapeutic exposure required for CMV prevention and treatment. Few reports directly address the incidence, consequences, and risk factors for hematologic toxicities related to ganciclovir therapy. Nevertheless, leukopenia, thrombocytopenia, and anemia occur in 5-50% of patients. Current strategies, focused on ganciclovir dose reduction, may increase the risk of CMV reactivation and drug-resistant disease. The current article reviews the incidence, risk factors, and consequences of ganciclovir-associated hematologic adverse events in transplant recipients. Management strategies, including ganciclovir dose reduction, and the addition of CMV hyperimmune globulin are discussed. Exposing this relatively frequently occurring, but uncommonly discussed, toxicity should lead to better avoidance and treatment strategies, without placing patients at increased risk of CMV disease.

Use of bivalirudin to prevent thrombosis following orthotopic liver transplantation in a patient with Budd-Chiari syndrome and a history of heparin-induced thrombocytopenia.

Type II heparin-induced thrombocytopenia (HIT) is an immune-mediated syndrome that may arise in a time-dependent manner following heparin therapy, placing patients at significant risk for thromboembolic events. Therapy includes anticoagulation with a direct thrombin inhibitor and avoidance of heparin. We report a patient with Budd-Chiari syndrome and a history of heparin-induced thrombocytopenia who presented for orthotopic liver transplant and required postoperative anticoagulation with bivalirudin. During the post-transplant graft function improvement, we observed a significant dose-effect alteration manifested by an increased bivalirudin dose requirement as factor V activity increased. This observation is an important consideration in the attempt to maintain an optimal balance between effective anticoagulation and a reduced risk of postoperative bleeding.

cAMP-specific phosphodiesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non-redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP cell signalling.

The PDE4 (phosphodiesterase-4) enzyme family consists of a distinct array of N-terminal splice variant isoforms arising from four subfamily genes (4A, 4B, 4C and 4D). These all hydrolyse specifically the intracellular second messenger cAMP. Although identical in catalytic function, each isoform appears to serve a non-superfluous regulatory role. For example, a beta-arrestin-sequestered subpopulation of the PDE4D5 isoform specifically regulates the phosphorylation of the beta(2)-AR (beta(2)-adrenergic receptor) by PKA (protein kinase A; also called cAMP-dependent protein kinase). This was elucidated by the use of novel technologies, including dominant-negative approaches, siRNA (small interfering RNA) knockdown and spot-immobilized peptide array analyses. Functional phenotypes uncovered using these methodologies have shown that beta-arrestin-sequestered PDE4D5 shapes the spatial cAMP gradient around the membrane-bound beta(2)-AR, regulating its phosphorylation by PKA and its ability to activate ERK (extracellular-signal-regulated kinase) through G(i) in cardiomyocytes and HEK-293 (human embryonic kidney)-B2 cells. This approach has provided the very first identification of a non-redundant and specific role for a PDE isoform. The fact that phenotypes can be uncovered by displacing PDE4 isoforms from specific anchor sites using dominant-negative constructs and cell-permeable peptides points to novel means for developing therapeutics aimed at disrupting specifically sequestered PDE isoforms and even specifically sequestered subpopulations of individual isoforms.