Active disease is independently associated with more severe anxiety rather than depressive symptoms in patients with systemic lupus erythematosus.

OBJECTIVE: The inter-correlation between and co-existence of depression and anxiety may engender inconsistency in addressing the relationship between the severity of depression and disease activity of systemic lupus erythematosus (SLE). We aimed at identifying whether lupus disease activity is independently associated with depression and anxiety in lupus patients. METHODS: Adult lupus patients were assessed for the severity of depressive and anxiety symptoms and lupus disease activity by using the Hospital Anxiety and Depression Scale (HADS) and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), respectively. Age- and gender-matched healthy controls (HCs) were recruited for comparison. Prevalence and severity of depressive and anxiety symptoms were compared between lupus patients and HCs. Independent relationships between the severity of anxiety (HADS-Anxiety) and depressive (HADS-Depression) symptoms, and SLEDAI were studied with regression models. RESULTS: In total, 110 lupus patients and 110 HCs were studied. Lupus patients had significantly higher HADS scores than HCs (10.82 ± 6.5 vs. 7.34 ± 4.9, p < 0.001). Significantly more lupus patients had anxiety (40.9 vs. 21.8%, p = 0.002) and depressive symptoms (15.5 vs. 6.4%, p = 0.025) than HCs. Multiple linear regression analyses revealed that SLEDAI (ß = 0.160, p = 0.016), calcineurin inhibitor non-use (ß = -1.929, p = 0.041) and past cyclophosphamide non-use (ß = -1.603, p = 0.039) independently predicted HADS-Anxiety amongst lupus patients even after adjusting for HADS-Depression. Conversely, SLEDAI (ß = 0.014, p = 0.834) lost its significant univariate correlation with HADS-Depression after controlling for HADS-Anxiety and other covariates. CONCLUSION: Anxiety is more common in lupus patients than in HCs, and its severity is independently associated with more active SLE regardless of the presence or absence of concomitant depression.

An integrative review of experiences of patients with rheumatoid arthritis.

AIM: To provide an overview of evidence on the experiences and needs of adults living with rheumatoid arthritis (RA). BACKGROUND: Many research have studied the impact of RA on one's quality of life, but no reviews have examined the overall experiences of patients living with RA. METHODS: An integrative review was performed to synthesize the experiences and/or needs of adult patients with RA based on articles retrieved from databases of CINAHL, Scopus, PubMed, PsycINFO and Web of Science, and published between January 2003 and March 2014 in English. RESULTS: A total of 38 studies were reviewed. RA has adverse effects on patients' quality of life due to its negative impacts in their physical and psychosocial aspects of health. Patients with RA cope with these impacts using various methods and are frequent users of healthcare services. However, few studies have evaluated patients' further needs in coping or the effectiveness of their coping mechanisms, and patients' experiences with health care. CONCLUSION: This review provides evidence for healthcare professionals to gain a better understanding of RA patients' experiences and needs. Future studies can explore interventions that will enhance the quality of current healthcare practices and ultimately improve patients' quality of life. IMPLICATIONS FOR NURSING AND HEALTH POLICY: Establishing a positive healthcare professional-patient relationship is crucial and healthcare professionals are in a position to provide greater informational and emotional support to patients. Policy makers and healthcare organizations need to look into ways to enhance the healthcare services to better suit RA patients' needs.

Infliximab in severe active ankylosing spondylitis with spinal ankylosis.

BACKGROUND: Infliximab is an anti-tumour necrosis factor monoclonal antibody, which significantly improves pain, stiffness and functional disability outcomes in patients with active ankylosing spondylitis. There are limited data available on the efficacy of this treatment for the subgroup with established spinal ankylosis. AIM: To compare the treatment response of infliximab in active severe ankylosing spondylitis for patients with and without radiographic evidence of spinal ankylosis in the clinical practice setting. METHODS: Twenty-seven patients with mean Bath Ankylosing Spondylitis Disease Activity Index of 8.7, all HLA-B27 positive, with 11 (41%) having spinal ankylosis, were studied for 54 weeks. The qualification for initial and ongoing infliximab treatment was defined by the Australian Pharmaceutical Benefit Schedule (PBS), and 5 mg/kg of infliximab was given at 0 week (baseline), repeated at 2 and 6 weeks and every 6 weeks thereafter. At each time point, PBS-mandated and international consensus response measures were completed. Disease activity and outcome measures for spinal ankylosis subgroup and those who did not have spinal ankylosis were cross-sectionally compared at baseline and 1 year. RESULTS: Patients with spinal ankylosis tended to be older (P = 0.01). Although the subgroup with spinal ankylosis had higher baseline activity scores, the only significant difference between the subgroups was the degree of morning stiffness (P = 0.04). By 54 weeks, all patients including the subgroup with spinal ankylosis fulfilled the PBS criteria for continuation of treatment. Majority of patients including the subgroup with spinal ankylosis achieved the various international consensus response measures. Patients with spinal ankylosis also experienced significant improvements in health-related quality of life, with majority returning to full-time employment by 1 year. CONCLUSION: In real-life clinical practice, patients with established disease with spinal ankylosis and high levels of inflammation and disease activity can achieve a major clinical response with infliximab.

Increasing podiatry referrals for patients with inflammatory arthritis at a tertiary hospital in Singapore: A quality improvement project.

BACKGROUND: Foot disease is highly prevalent in people with inflammatory arthritis and is often under-recognized. Podiatry intervention can significantly reduce foot pain and disability, with timely access being the key factor. The aim of this study was to plan and implement a quality improvement project to identify the barriers to, and improve, uptake of podiatry services among patients with inflammatory arthritis-related foot problems seen at a tertiary hospital in Singapore. METHOD: A 6-month quality improvement program was conducted by a team of key stakeholders using quality improvement tools to identify, implement and test several interventions designed to improve uptake of podiatry services. The number of patients referred for podiatry assessment was recorded on a weekly basis by an experienced podiatrist. The criterion for appropriate referral to podiatry was those patients with current or previous foot problems such as foot pain, swelling and deformity. RESULTS: Interventions included education initiatives, revised workflow, development of national guidelines for inflammatory arthritis, local podiatry guidelines for the management of foot and ankle problems, routine use of outcome measures, and introduction of a fully integrated rheumatology-podiatry service with reduced cost package. Referral rates increased from 8% to 11%, and were sustained beyond the study period. Complete incorporation of podiatry into the rheumatology consultation as part of the multidisciplinary team package further increased referrals to achieve the target of full uptake of the podiatry service. CONCLUSION: Through a structured quality improvement program, referrals to podiatry increased and improved the uptake and acceptance of rheumatology-podiatry services.

Prevalence of foot problems in people with inflammatory arthritis in Singapore.

BACKGROUND: Foot problems are highly prevalent in people with inflammatory arthritis reported from studies in the UK, Europe and New Zealand, but there is limited evidence from Southeast Asia. The study aim was to evaluate the prevalence of foot problems in people with inflammatory arthritis in Singapore. METHODS: People with inflammatory arthritis were recruited from the rheumatology outpatient clinic of a tertiary hospital in Singapore. Disease and clinical characteristics included age, sex, disease duration, current blood tests and medications. The Leeds Foot Impact Scale was used to evaluate foot impairment/disability and the Modified Health Assessment Questionnaire was used to assess global function. RESULTS: We recruited 101 people with inflammatory arthritis, of which 50 % were female. The majority of participants were Chinese (70 %). The mean (SD) age was 52 (15) years, and the mean (SD) disease duration was 9.3 (0.3) years. The most commonly reported inflammatory arthritic conditions were rheumatoid arthritis (46), gout (31) and spondyloarthritis (15 %). The mean (SD) of the total Leeds Foot Impact Scale was 17 (13) indicating moderate to severe levels of foot impairment and activity limitation. Over 80 of participants reported foot pain during the course of their condition, and 48 % reported current foot pain. Despite the high prevalence of foot pain, only 21 participants (21 %) had been referred to a podiatrist. CONCLUSION: This is the first study to investigate the prevalence of foot problems in people with inflammatory arthritis from Singapore. The majority of the participants reported foot problems, but had not been referred to a podiatry service.

Footwear characteristics in people with inflammatory arthritis in Singapore.

BACKGROUND: Foot problems are common in people with inflammatory arthritis. Despite suitable footwear having the potential to alleviate pain, improve mobility and maintain independence, previous studies have found many people with inflammatory arthritis wearing poorly fitting and inappropriate footwear. Footwear styles and characteristics have not been reported in a Singapore inflammatory arthritis population. The objective of this study was to identify current footwear styles and characteristics of footwear worn by people with inflammatory arthritis in Singapore. METHODS: One-hundred-and-one participants with inflammatory arthritis were recruited from the rheumatology outpatient clinic of a large public hospital in Singapore. Disease and clinical characteristics were recorded. A patient-reported outcome included current foot pain. An objective footwear assessment of style, age of shoe, fit and construction was conducted. RESULTS: The majority of participants were Chinese women with a mean (SD) age was 52.0 (15.0) years old and a mean (SD) disease duration of 9.3 (0.3) years. We found 50 % of participants (n = 51) reported footwear problems. Sandals (n = 27, 26 %), flip-flops (n = 19, 19 %) and moccasin type (n = 19, 19 %) was the most common footwear choice. Evaluation of footwear characteristics found that there was a lack of motion control features. Only 32 (32 %) participants had correctly fitting footwear with regard to length, width and depth. No participant was wearing therapeutic footwear. CONCLUSION: This study provides the first insight into footwear preferences of people with inflammatory arthritis in Singapore. Use of slip-on and poorly fitting footwear was found to be common in people with inflammatory arthritis. Further research on footwear preferences in Southeast-Asian communities needs to take into account cultural habit and preference, socio-economic status, footwear options and affordability.

Constructing Kinetic Network Models to Elucidate Mechanisms of Functional Conformational Changes of Enzymes and Their Recognition with Ligands.

Enzymes are biological macromolecules that catalyze complex reactions in life. In order to perform their functions effectively and efficiently, enzymes undergo conformational changes between different functional states. Therefore, elucidating the dynamics between these states is essential to understand the molecular mechanisms of enzymes. Although experimental methods such as X-ray crystallography and cryoelectron microscopy can produce high-resolution structures, the detailed conformational dynamics of many enzymes still remain obscure. While molecular dynamics (MD) simulations are able to complement the experiments by providing structure-based dynamics at atomic resolution, it is usually difficult for them to reach the biologically relevant timescales (hundreds of microseconds or longer). Kinetic network models (KNMs), in particular Markov state models (MSMs), hold great promise to overcome this challenge because they can bridge the timescale gap between MD simulations and experimental observations. In this chapter, we review the procedure of constructing KNMs to elucidate the molecular mechanisms of enzymes. First, we will give a general introduction of MSMs, including the methods to construct and validate MSMs. Second, we will present the applications of KNMs to study two important enzymes: the human Argonaute protein and the RNA polymerase II. We conclude by discussing the future perspectives regarding the potential of KNMs to investigate the dynamics of enzymes' functional conformational changes.

Utility of the Morisky Medication Adherence Scale in gout: a prospective study.

BACKGROUND: The outcomes of any chronic illness often depend on patients' adherence with their treatment. A tool is lacking to assess adherence in gout that is standardized, allows real-time feedback, and is easy to understand. OBJECTIVE: We set out to evaluate the utility of the 8-item Morisky Medication Adherence Scale (MMAS-8) in monitoring medication adherence in a multiethnic Asian gout cohort on urate-lowering therapy (ULT). METHODS: This cohort study recruited patients with gout where baseline and 6-monthly clinical data, self-report of adherence, and health status by Gout Impact Scale (GIS) and EuroQoL-5 dimension 3 levels were collected. Those who received at least 9 months of ULT were analyzed. Convergent and construct validities of MMAS-8 were evaluated against medication possession ratio (MPR) and known groups, clinical outcomes, and patient-reported outcomes. Internal consistency and test-retest reliability were assessed using Cronbach's alpha and intraclass correlation coefficient (ICC), respectively. RESULTS: Of 91 patients, 92.3% were male, 72.5% Chinese with mean age 53.5 years. MMAS-8 (mean 6.17) and MPR (mean 96.3%) were poorly correlated (r=0.069, P=0.521). MMAS-8 did not differ between those who did or did not achieve target serum urate (SU) <360 µmol/L (P=0.852); or among those whose SU improved, stagnated, or worsened during follow-up (P=0.777). Adherence was associated with age (ß=0.256, P=0.015) and education level (P=0.011) but not comorbidities, polypharmacy, or flare frequency. Concerns for medication side effects and anxiety or depression were associated with lower MMAS-8 (P<0.005). Internal consistency was acceptable (α=0.725) and test-retest reliability was satisfactory (ICC =0.70, 95% confidence interval [CI] 0.36-0.88). CONCLUSION: MMAS-8 had limited construct validity in assessing medication adherence to ULT in our gout patients. Nevertheless, it identified patients bothered or worried about ULT side effects, and those with underlying anxiety or depression, for whom targeted education and coping support may be useful.

Silastic medialization for unilateral vocal fold palsy.

From August 1994 to September 1995, 12 silastic medialization procedures were performed for the treatment of vocal fold palsy. The causes included tuberculous fibrosis, carcinoma of the bronchus, post-oesophagectomy for carcinoma of the oesophagus and idiopathy. Early operation was performed in cases due to malignant conditions to relieve symptoms. In those with benign conditions, operation was performed if conservative treatment failed to control the symptoms in six months. The efficacy of silastic medialization for the treatment of dysphonia was evaluated by both subjective and objective voice assessment. The results indicate that the procedure is effective in the relief of dysphonia in unilateral vocal fold palsy. Only one patient in the study required a revision operation due to unsatisfactory voice quality. The procedure has the advantages of being tunable, reversible, and suitable for old and debilitated patients. The long term benefits of the procedure require further study.

Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population.

UNLABELLED: We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. IMPORTANCE: The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.

What are the best markers for disease progression in osteoarthritis (OA)?

To be able to prevent progression of osteoarthritis, the knowledge of prognostic factors of this progression is important. If certain prognostic factors are modifiable, they may enhance our ability to reduce osteoarthritis progression. Even if these prognostic factors are not modifiable, they can still be used to identify high-risk groups, which may have implications for patient information and the perspective of medical treatment. Prognostic factors of progression are reviewed here, mainly for hip and knee osteoarthritis as most data available concern these localisations. Areas of further research are highlighted.

Beyond demographic transition: industrialization and population change in Singapore.

PIP: This paper discusses the demographic basis on which Singapore's population policy is founded. It examines the factors leading to rapid fertility decline and discusses the incentives to promote higher fertility and their prospects for success. In 1986, Singapore's total fertility rate was 1.44, the lowest in Asia. The decline is equally sharp for all ethnic groups. If this rate remains constant, Singapore's population would peak at 2.9 million in 2015 and then begin to decline. In 1957, the mean age at marriage was age 20; by 1986, it had increased to age 30. This delay in marriage is associated with increased education. The mean age of mothers at 1st birth has risen steadily from 23.4 years in 1970 to 26.7 years in 1986. As a result of the liberalization of access to abortion in 1973, there has been a steady increase in the number of abortions in Singapore. Female sterilization has become increasingly prevalent over the years. The government's announcement of a new population policy came as no surprise since fertility trends had been discussed by ministers. The new policy slogan "Have 3, and More If You Can Afford It" reflects the government's intention of promoting the 3-child family. A special tax rebate of $S20,000 is given to parents for the birth of their 3rd child after 1 January 1987. Children from 3-child families are given priority in primary school registration. Delivery and hospital costs arising from the delivery of the 4th child can be offset against the parents' earned income up to a maximum of $S3000. Priority allocation and other incentives are given to families who want to upgrade their Housing and Development Board apartments after the birth of their 3rd child. Married female civil servants are able to enjoy special unrecorded leave to look after their sick children who are under 6 years old. A unit has been established in the government to promote interaction among male and female university graduates. A public education program on the 3-child norm will be launched. Demographic trends suggest that a rebound of the fertility rate is unlikely to occur on its own. To the extent that small family size is rationally compatible with modern society, any effort to promote larger family size, however intense, is bound to have modest results.^ieng

Micro-consequences of low fertility in Singapore.

PIP: When below-replacement fertility was reached in Singapore due to the decline in female marriage rates and the reduction in average family size, economic resources were no longer needed to support a dependent population. Funds were used for developmental projects which increased per capita income and the economic base. But manpower needs were unmet because the younger cohort size was too small and the population was aging. Besides these changes in age and economic structure, some of the impact on family and occupational structure is described. For instance, there was a delay in marriage age from 23.7 years in 1970 to 26 years in 1989. This means daughters remain living with parents in Chinese and Indian households and in Malay households were this has been the practice. Consequently, daughters prevent married sons from sharing a parental home. Daughters assume the responsibilities for parental care. It is suggested that if inheritance patterns change to reflect a greater inheritance to daughters, daughters may choose greater independence from parental control. Social service for single women may require more flexibility and acceptance of lifestyles of single women living along. The smaller family size of 2.6 children also means a contraction of the childbearing period to age 35. Women 25-29 years in the labor force in 1989 constituted 74% and 50.2% for ages 40-44 with an overall rate of 48.4%. Rising educational attainment and opportunities available created this situation. With the changes in work priority comes the change in child care arrangements such that grandparents and relatives (30% in 1989) were used. Demand has increased for day care centers. Foreign household domestics are being recruited (7% of all households). Husband and wife relationships have placed the women in greater control over decision making particularly on the number of children. Daughters are not as discriminated against because of the economic returns. The rising status is reflected in educational attainment where 61% in 1989 have a secondary education or higher. Economic changes in the labor supply created a need for 10-15% of foreign workers in all sectors but not to exceed 50%. Levies on foreign workers were introduced to deter overreliance. Women have expanded most in clerical positions. It is expected that the 20-24 year cohort will continue to decline affecting the labor shortage.^ieng

Chromosomal mapping of human kininogen gene (KNG) to 3q26----qter.

The structural gene for human kininogen (KNG) was localized to chromosome 3q26----qter by in situ hybridization. The assignment substantiates the evolutionary relationship of kininogen to two other members of the cystatin superfamily, alpha-2-HS-glycoprotein and histidine-rich glycoprotein, which also map to chromosome 3.

Partial characterization of the active site human platelet cAMP phosphodiesterase, PDE3A, by site-directed mutagenesis.

Phosphodiesterases (PDE) are important for the downregulation of the intracellular level of the second messenger cyclic adenosine monophosphate (cAMP) by hydrolyzing cAMP to 5'AMP. Previous studies from our laboratory suggested that the human platelet PDE3A active site has two essential histidine residues and one cysteine residue. We therefore decided to begin mutating histidines and cysteines in the conserved domain of PDE3A. A truncated but catalytically active recombinant PDE3A protein was expressed in yeast cells. A six-histidine tag was engineered to the carboxyl end to facilitate protein purification by nickel column. This cDNA construct, PDE3ADelta1, was used for the mutant construction. Mutations were introduced by mutant oligonucleotides during PCR or DNA replication and were confirmed by sequencing. The mutant cDNAs were expressed in PDE-deficient yeast host Saccharomyces cerevisiae strain GL62. The expression levels of the recombinant PDE mutant proteins were monitored by Western blotting using a rabbit anti-platelet PDE3A polyclonal antibody. The kcat, KM, and IC50 for cyclic guanosine monophosphate (cGMP) and IC50 for milrinone were determined for each mutant. Two highly conserved histidines and four conserved cysteines were each mutated to alanine. C816 is present in the 44-amino acid insert unique to PDE3. The mutant C816A is poorly expressed (>2%) and probably is not folded appropriately. H840 is the second histidine in the second motif for metal binding, HDXXH. The mutation H840A, although expressed moderately well, has undetectable activity and is probably responsible for binding a bivalent cation, e.g., Mn2+, essential for catalysis. H869 is a highly conserved amino acid not in a metal-binding motif. H869A is well expressed with a normal kcat. However, the Km for cAMP and the IC50 for cGMP are each fourfold greater in the mutant than those in the wild-type recombinant, suggesting that this histidine is in the inhibitory binding site. Three well-conserved cysteines were mutated, but C942A, C945A, and C1013A all had kinetic values similar to the wild type. The results have identified a histidine at the active site essential for catalysis and a second histidine important for inhibitor binding and have further supported the essential nature of the unique 44-amino acid insert.

Genetic basis of total kininogen deficiency in Williams' trait.

High and low molecular weight kininogens (HK and LK) modulate inflammatory responses, serve as precursors of kinins, inhibit cysteine proteinases, and modulate thrombin activation of platelets. Differential splicing yields two different mRNAs for HK and LK. We report the first molecular characterization of a kininogen gene mutation in a patient lacking LK and HK. No gross DNA deletion or insertion was detected by Southern blots, and LK and HK mRNAs were normal size on Northern blots. Exon sequences amplified by polymerase chain reaction showed a C-->T transition at nucleotide 587, resulting in a CGA (Arg)--> TGA (Stop) mutation in exon 5 before the splice site in exon 10, thus preventing synthesis of both HK and LK. The mutation eliminated the recognition site of the restriction enzyme Csp45I. Results of Csp45I digestion of the polymerase chain reaction-amplified exon 5 DNA fragment of the patient (lacking kininogens), three daughters (approximately 50% HK), and 1 granddaughter (normal HK) revealed that the patient was homozygous, while the three daughters were heterozygous, and the granddaughter was normal. We conclude that a single base mutation in the kininogen gene exon 5 was responsible for kininogen deficiency in the Williams family.

Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis.

Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.

Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells.

Prostaglandins inhibit platelet activation by stimulating intracellular cyclic AMP formation. We have postulated that intracellular cyclic AMP levels in platelets are buffered by a distinct prostaglandin receptor that mediates inhibition of cyclic AMP formation. In order to provide evidence for the model, we have cloned the cDNA coding for a prostaglandin receptor EP3 subtype, which is coupled to inhibition of adenylate cyclase, from the megakaryocytic cell line human erythroleukaemia (HEL) cells. A PCR-generated hybridization probe, produced using primers based on the sequence of the mouse prostaglandin EP3 receptor published by Sugimoto, Namba, Honda, Hayashi, Negishi, Ichikawa and Narumiya [(1992) J. Biol. Chem. 267, 6463-6466], was used to screen a lambda gt11 HEL cell cDNA library. The composite full-length cDNA clone HEP3, generated from the two partial clones pHEP3-7 and pHEP3-5, is 1.6 kb long with an open reading frame coding for 390 amino acids. This clone is 83% identical to the alpha subtype of the mouse EP3 receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP3 subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E2 (PGE2) and binding PGE2 with high specificity and a Kd of 3.2 nM. Radiolabelled PGE2 could be displaced by prostaglandins in the order PGE2 = PGE1 > iloprost = PGD2. Northern blot analysis revealed that the receptor is also present in human kidney.

Candidate reference method for determination of total bilirubin in serum: development and validation.

This candidate Reference Method for measuring total bilirubin in serum is based on the Jendrassik-Gróf principle (Clin Chem 29: 297-301, 1983). Standard Reference Material no. 916 bilirubin (National Bureau of Standards) is used as the standard. Bilirubin standard solutions may be prepared either in human serum or in 40 g/L albumin solution (human or bovine), because we found the molar absorptivity of the azopigment at 598 nm to be identical in these media. The absorptivities of the unconjugated and conjugated azopigments appear to be identical, but the conjugated azopigment is completely hydrolyzed in the final reaction mixture. Bilirubin added to serum from adults or neonates was quantitatively accounted for. Interference by hemoglobin (up to 2 g/L), ascorbic acid (up to 20 mg/L), or zinc (at physiological concentrations) is negligible. Of the therapeutic drugs we tested, only L-dopa and alpha-methyldopa interfere. We established normal adult reference values for total bilirubin and examined the intraindividual variation in 19 subjects.