Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

Background and purpose:

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A_2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine.

Experimental approach:

The trafficking of the wild type A_2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.

Key results:

The wild type A_2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln³²⁵-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu³³⁰-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln³²⁵-stop, Ser³²⁶-stop and Phe³²⁸-stop receptors. Following internalization, the wild type A_2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln³²⁵-stop receptor did not recycle.

Conclusions and implications:

Deletion of the COOH-terminus of the A_2B adenosine receptor beyond Leu³³⁰ switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A_2B adenosine receptor following prolonged agonist addition.     

Ex vivo expansion of megakaryocyte precursor cells in autologous stem cell transplantation for relapsed malignant lymphoma

To evaluate the impact of ex vivo expanded megakaryocyte (MK) progenitors on high-dose chemotherapy-induced thrombocytopenia, we conducted a phase II study in 10 patients with relapsed lymphoma. Two fractions of peripheral blood progenitor cells (PBPC) were cryopreserved, one with enough cells for at least 2 x 10(6) CD34+ cells/kg and a second obtained after CD34+ selection. Ten days before autologous stem cell transplantation, the CD34+ fraction was cultured with MGDF+SCF for 10 days. After BEAM (BCNU, cyclophosphamide, cytarabine, and melphalan) chemotherapy, patients were reinfused with standard PBPC and ex vivo expanded cells. No toxicity was observed after reinfusion. The mean fold expansion was 9.27 for nucleated cells, 2 for CD34+ cells, 676 for CD41+ cells, and 627 for CD61+ cells. The median date of platelet transfusion independence was day 8 (range: 7-12). All patients received at least one platelet transfusion. In conclusion, ex vivo expansion of MK progenitors was feasible and safe, but this procedure did not prevent BEAM-induced thrombocytopenia. Future studies will determine if expansion of higher numbers of CD34+ cells towards the MK-differentiation pathway will translate into a functional effect in terms of shortening of BEAM-induced thrombocytopenia.     

Haplotype association analysis of the polymorphisms Arg16Gly and Gln27Glu of the adrenergic beta2 receptor in a Swedish hypertensive population

The Arg16Gly and the Gln27Glu polymorphisms in the gene for the beta2-adrenergic receptor (beta2AR) have been linked to an increased risk for cardiovascular disease. The aim of the present study was to evaluate the significance of these haplotypes for development of myocardial infarction (MI) as well as other cardiovascular phenotypes. In a prospective study cohort (CAPPP), 522 hypertensive patients (174 MI and 348 matched controls) were analysed for the Arg16Gly and the Gln27Glu polymorphisms by dynamic allele-specific hybridisation. The haplotype could successfully be determined in 516 patients. Haplotype was not significantly associated with MI. Systolic blood pressure (SBP) was higher in patients with Arg16Gly+Gln27Gln and lower in patients with Arg16Gly+Gln27Glu as compared with the other haplotypes. Haplotype was not associated with body mass index, diastolic blood pressure, cholesterol, LDL, HDL triglycerides or a diagnosis of diabetes mellitus. The present study found no evidence that haplotype for the two most common polymorphisms in the beta2AR are associated with development of MI in a Swedish hypertensive population, but haplotype may be associated with SBP.     

Acute exercise-induced nitric oxide production contributes to upregulation of circulating endothelial progenitor cells in healthy subjects

Exercise has been proved to promote the number and activity of circulating endothelial progenitor cells (EPCs) in humans, which contributes to improvement in endothelial function and maintenance of cardiovascular homoeostasis. However, the mechanism underlying the effect of exercise on circulating EPCs in healthy subjects is not completely understood. Here, we investigated whether the regulation of acute exercise on circulating EPCs is associated with nitric oxide (NO), vascular endothelial growth factors (VEGF) and granulocyte macrophage colony stimulating factor (GM-CSF) known to modulate circulating EPCs in healthy subjects. A total of 16 healthy male volunteers underwent a modified Bruce treadmill acute exercise protocol. The number and activity of circulating EPCs, as well as the levels of NO-VEGF and GM-CSF in plasma and culture medium before and after exercise in healthy subjects were measured. The number and activity of circulating EPCs after acute exercise were significantly higher than those before exercise in healthy subjects. In parallel, acute exercise significantly enhanced plasma NO level in healthy subjects. There is a significant linear regression relationship between the enhanced plasma NO level and increased number or activity of circulating EPCs. However, no change of plasma VEGF and GM-CSF level was observed after acute exercise. The secretion of NO-VEGF and GM-CSF by cultured EPCs remained unchanged in response to acute exercise. The present study demonstrates for the first time that acute exercise-induced NO production contributes to upregulation of circulating EPCs in healthy subjects, which suggests that NO plays an important role in the regulation of exercise on circulating EPCs.     

Syndrome X in 8-y-old Australian children: stronger associations with current body fatness than with infant size or growth

OBJECTIVE: Syndrome X (clustering of insulin resistance, dyslipidaemia and hypertension) in adults with central obesity has been suggested to be a consequence of poor foetal development. We investigated clustering of syndrome X factors in a sample of 8-y-old Australian children, and whether the clusters were associated with size at birth and childhood obesity. DESIGN: Longitudinal, 1997 follow-up of children enrolled as singleton-born neonates in 1989. SUBJECTS: A total of 298 healthy Australian children (208 boys, 90 girls, age range 7.4-8.9 y). MEASUREMENTS: Anthropometry at birth and at 4 weeks. In 1997, at 8 y of age: fasting insulin and glucose, total and HDL-cholesterol, triglycerides and blood pressure. RESULTS: Adverse levels of insulin and glucose, cholesterol and triglycerides co-existed more often than expected by chance (P<0.01). Three factors were identified in factor analysis: one loading on systolic and diastolic blood pressure ('blood pressure'); a second loading on insulin and glucose ('insulin resistance'); and a third loading negatively on HDL-cholesterol and positively on triglycerides ('dyslipidaemia'). The blood pressure factor was correlated with fatness at age 8 y (eg fat mass estimated from skin folds, r=0.11) and, after adjustment for current size, with birth weight (r=-0.15). Fat mass was also correlated with both 'insulin resistance' (r=0.24) and 'dyslipidaemia' (r=0.19). The increase in 'insulin resistance' (P=0.03) and 'dyslipidaemia' (P<0.01) per category of fat mass was greatest for subjects with higher-than-median subscapular-to-triceps ratio of skin folds. Neither 'insulin resistance' nor 'dyslipidaemia' was associated with anthropometry at birth. CONCLUSIONS: The Syndrome X risk variables clustered among children who had a tendency to deposit fat on the trunk. There was no evidence in this sample that infant size predicts development of the insulin resistance or dyslipidaemic components of the syndrome by age 8.     

Necrotic myocarditis in acute eosinophilic lymphoblastic leukaemia

INTRODUCTION: Hypereosinophilia can cause severe cardiac complications. The association between an acute lymphoblastic leukemia and hypereosinophilia was rare. We report a case of a 29-year-old man who presented a heart failure secondary to necrotic myocarditis related to an acute eosinophilic lymphoblastic leukaemia. EXEGESIS: The patient developed a heart failure and secondary a cardio-embolic stroke, due to a large mobile left ventricle thrombosis. His peripheral blood showed a total white count of 28,500 leucocytes/mm3 with 18,800 eosinophils/mm3. The myelogram cytology showed precursor B-cell acute lymphoblastic leukaemia with hypereosinophilia. CONCLUSION: The possibility of the rapid emergence of cardiac lesions in hypereosinophilic syndromes warrants very close physician vigilance. An Echocardiography and MRI performed at the early stage and in the follow-up allow to detect and to manage these cardiac disorders.     

Interstitial lung disease due to domestic moulds

Identifying the role of fungi present in the domestic environment in the development of interstitial pneumonia can be a difficult clinical problem. We report a case of interstitial lung disease case occurring in a 53-year-old patient. He presented with profound hypoxemia (PaO(2) 54mmHg). Chest CT showed diffuse ground glass opacities. Initial blood tests for allergy and autoimmune disease were negative. Faced with a worsening of his clinical status after returning home he was hospitalized several times. At fibreoptic bronchoscopy, multiple white deposits were observed. Bronchoalveolar lavage with differential cell count was performed, revealing a 23% lymphocytosis. Serology for specific household molds showed moderate reaction to various molds found in homes, especially Stachybotrys?chartarum. Pulmonary function tests revealed a moderate restrictive pattern with impaired diffusion of carbon monoxide and a bronchiolocentric interstitial pneumonia was found at lung biopsy. After a permanent move to a new residence, clinical parameters, radiological, biological and functional normalized. The final diagnosis was interstitial lung disease related to mycotoxins of S.?Chartarum. The diagnosis of hypersensitivity pneumonitis to domestic mold or interstitial lung disease secondary to mycotoxins should be considered in patients presenting with interstitial pneumonia and requires specific investigations to ensure that an environmental cause with an allergic or toxic role is not missed.     

Patient evaluation of the care and rehabilitation process in geriatric hospital care

Purpose : To gain a deeper understanding of how elderly persons experience and evaluate the care and rehabilitation process.

Method : Qualitative interview data from elderly patients were analysed using a grounded theory approach. The patients were interviewed twice, at the beginning of geriatric hospital care and some weeks after discharge.

Results : The patient-perceived outcome of the care and rehabilitation process reflected two dimensions; the effect on their health and the quality of the process, i.e. how their needs were met. The analysis revealed that the patients' needs differed during the care and rehabilitation process. It also indicated that patients perceived their needs and the care differently based on their previous experience of the care unit, their perceived trajectory of illness and their 'patient character' which represented the patient's definition of himself/herself and the situation. A hypothetical model of the patients' evaluation process has been derived.

Conclusion : The results indicate the importance of using a process perspective in the assessment and the interpretation of patient-perceived outcome of care and rehabilitation, and that patient expectations, trajectories of illness and the patient character must be taken into consideration.     

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG → GAG, and P854L, CCG → CTG) and, additionally, the mutation: G714R, GGG → AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA → AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughter's red cell membrane.