The C-terminal kinase domain of the p34cdc2-related PITSLRE protein kinase (p110C) associates with p21-activated kinase 1 and inhibits its activity during anoikis.

The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases. During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C). The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells. In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells. To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C. The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis. The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1. Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed. Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C. Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis. Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.

Mice lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in multiple regions of the central nervous system

Ataxia with vitamin E deficiency is caused by mutations in a-tocopherol transfer protein (a-TTP) gene and it can be experimentally generated in mice by a-TTP gene inactivation (a-TTP-KO). This study compared a-tocopherol (a-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and a-TTP-KO mice. All brain regions of female WT mice contained significantly higher a-T than those from WT males. a-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain a-T concentrations do not appear to be determined by a-TTP expression which was undetectable in all brain regions. All the brain regions of a-TTP-KO mice were severely depleted in a-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of a-TTP-KO mice. The results show that both gender and the hepatic a-TTP, but not brain a-TTP gene expression are important in determining a-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in a-TTP-KO mice in spite of the severe a-tocopherol deficiency in the brain starting at an early age.

Quantification of Hox and Surfactant Protein-B Transcription during Murine Lung Development.

Abstract BACKGROUND: Genetic processes underlying fetal lung development and maturation are incompletely understood. Better knowledge of these processes would provide insights into the causes of lung malformations and prevention of respiratory distress syndrome and the potential adverse effects of glucocorticoids. Hox genes are involved in the lung branching morphogenesis and maturation of respiratory epithelium, but their expression pattern remains to be defined. OBJECTIVES: We hypothesized that genes involved in lung branching would be downregulated during early development, whereas those involved in maturation would be unchanged or upregulated. METHODS: TaqMan real-time primers and probes were designed for all 39 murine Hox genes, and the murine SP-B gene and transcription profiles of these genes were obtained from whole lungs isolated at e14.5, e16.5, e18.5, e19.5 and postnatal days 1 and 20. RESULTS: Hox genes in clusters A and B, specifically those between paralog groups 3 and 7, were the most represented, with Hoxa4 and Hoxa5 being the most highly transcribed. A wave of reduced transcription in 16 Hox genes, coincident with increased SP-B transcription, was observed with advancing gestation. Consistently high transcription of Hoxa5 from e14.5 to postnatal day 20 may indicate that sustained transcription is required for normal lung maturation. When e15.5 lungs were cultured with dexamethasone, Hoxb6, Hoxb7 and Hoxb8 levels were significantly upregulated, creating the potential for modulation of diverse downstream target genes. CONCLUSIONS: Improved understanding of the genetic processes underlying lung development afforded by our Q-PCR platform may allow development of more specific methods for inducing fetal lung maturation.

High-dose adenosine overcomes the attenuation of myocardial perfusion reserve caused by caffeine.

Objectives:
We studied whether an increase in adenosine dose overcomes caffeine antagonism on adenosine-mediated coronary vasodilation.

Background:
Caffeine is a competitive antagonist at the adenosine receptors, but it is unclear whether caffeine in coffee alters the actions of exogenous adenosine, and whether the antagonism can be surmounted by increasing the adenosine dose.

Methods:
Myocardial perfusion scintigraphy (MPS) was used to assess adenosine-induced hyperemia in 30 patients before (baseline) and after coffee ingestion (caffeine). At baseline, patients received 140 µg/kg/min of adenosine combined with low-level exercise. For the caffeine study, 12 patients received 140 µg/kg/min of adenosine (standard) and 18 patients received 210 µg/kg/min (high dose) after caffeine intake (200 mg). Myocardial perfusion was assessed semiquantitatively and quantitatively, and perfusion defect was characterized according to the presence of reversibility.

Results:
Caffeine reduced the magnitude of perfusion abnormality induced by standard adenosine as measured by the summed difference score (SDS) (12.0 ± 4.4 at baseline vs. 4.1 ± 2.1 after caffeine, p < 0.001) as well as defect size (18% [3% to 38%] vs. 8% [0% to 22%], p < 0.01), whereas it had no effect on the abnormalities caused by high-dose adenosine (SDS, 7.7 ± 4.0 at baseline vs. 7.8 ± 4.2 after caffeine, p = 0.7). There was good agreement between baseline and caffeine studies for segmental defect category (kappa = 0.72, 95% confidence interval: 0.65 to 0.79) in the high-dose group. An increase in adenosine after caffeine intake was well tolerated.

Conclusions:
Caffeine in coffee attenuates adenosine-induced coronary hyperemia and, consequently, the detection of perfusion abnormality by adenosine MPS. This can be overcome by increasing the adenosine dose without compromising test tolerability.

From Rights to Compassion: The D Case and Contemporary Abortion Politics

This chapter is concerned with exploring the dynamics of contemporary debate on women’s reproductive choices and rights in the somewhat transformed social, political and economic context of the Republic of Ireland. News coverage of the events of April and May 2007 provide the focus of attention, as the case of ‘D’, a 17 year old in the temporary care of the state, seeking to terminate her pregnancy after a diagnosis of severe foetal abnormality, became yet again a focus of public debate on abortion access within the state. The analysis explores how the issues this case raised were framed in the public domain, in order to consider the shifting moral grammar shaping the debate. The paper explores the ways in which this case illustrates the ongoing tensions between changing characterisations of Irishness, and the social dynamics of access to reproductive rights, particularly for national minors in the care of the state.

Runx2, p53, and pRB Status as Diagnostic Parameters for Deregulation of Osteoblast Growth and Differentiation in a New Pre-Chemotherapeutic Osteosarcoma Cell Line (OS1)

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS I have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS I had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio, (P

Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma

To understand the molecular etiology of osteosarcoma, we isolated and characterized a human osteosarcoma cell line (OS1). OS1 cells have high osteogenic potential in differentiation induction media. Molecular analysis reveals OS1 cells express the pocket protein pRB and the runt-related transcription factor Runx2. Strikingly, Runx2 is expressed at higher levels in OS1 cells than in human fetal osteoblasts. Both pRB and Runx2 have growth suppressive potential in osteoblasts and are key factors controlling competency for osteoblast differentiation. The high levels of Runx2 clearly suggest osteosarcomas may form from committed osteoblasts that have bypassed growth restrictions normally imposed by Runx2. Interestingly, OS1 cells do not exhibit p53 expression and thus lack a functional p53/p21 DNA damage response pathway as has been observed for other osteosarcoma cell types. Absence of this pathway predicts genomic instability and/or vulnerability to secondary mutations that may counteract the anti-proliferative activity of Runx2 that is normally observed in osteoblasts. We conclude OS1 cells provide a valuable cell culture model to examine molecular events that are responsible for the pathologic conversion of phenotypically normal osteoblast precursors into osteosarcoma cells.

Preconception care for women with diabetes: the midwife's role

Diabetes mellitus is a chronic illness which affects a significant number of childbearing women. Despite the potential for adverse consequences for both maternal and fetal wellbeing, few women with diabetes plan their pregnancies to ensure that they enter pregnancy in optimal health. Furthermore, whilst adverse pregnancy outcomes are well documented for women with type I diabetes, it is now apparent that an increasing number of women with type II diabetes are becoming pregnant with similar adverse associated risk. There is an increasing recognition that significant adverse pregnancy outcomes are determined prior to a woman initiating pregnancy care, many of which could be minimised with the introduction of preconception care. As formalised preconception care clinics remain scant across the United Kingdom, there is an urgent need to increase the opportunities for the provision of preconception care and advice to women with diabetes. Midwives are ideally placed to provide preconception advice to women and could provide the missing link in terms of preconception advice for women with diabetes.

Natural history of idiopathic erythrocytosis - A retrospective case series review

Idiopathic Erythrocytosis (IE) is a diagnosis given to patients who have an absolute erythrocytosis (red cell mass more than 25% above their mean normal predicted value) but who do not have a known form of primary or secondary erythrocytosis (BCSH guideline, 2005). We report here the results of a follow-up study of 80 patients (44 male and 36 female) diagnosed with IE from the United Kingdom and the Republic of Ireland over a 10 year period. Baseline information was initially collected when investigating for molecular causes of erythrocytosis in this group. The diagnosis of IE was made on the basis of a raised red cell mass >25% above mean normal predicted value, absence of Polycythaemia Vera (PV) based on the criteria of Pearson and Messinezy (1996), and the exclusion of secondary erythrocytosis (oxygen saturation >92% on pulse oximetry, no history of sleep apnoea, no renal or hepatic pathology, and a normal oxygen dissociation curve (if indicated). The average age at diagnosis of erythrocytosis was 34.5 (2–74 years). Erythropoietin levels were available for 77/80 of the patients and were low in 18 (23%) and normal or high in 59 (74%). Ultrasound imaging was carried out in 67 patients (84%) at time of diagnosis and no significant abnormalities found. Fourteen patients had a family history of erythrocytosis. These patients have now been followed up for an average of 9.4 years (range 1–39). Out of 80 patients 56 patients can still be classified as having IE, of whom 52 are living (cause of death in the other 4 - lung cancer, RTA, sepsis, unknown). Thirty-five of these patients are regularly venesected, 3 take hydroxyurea (one also venesected), 11 receive no treatment while treatment is unknown in 2. Twenty take aspirin, 1 warfarin and 31 no thromboprophylaxis. Four of these patients had suffered thromboembolic complications (3 with CVA/TIAs and 1 with recurrent DVT) at or before their original diagnosis. Since diagnosis 8 patients have had 9 thrombotic events of which 7 were arterial (1 CVA, 3 TIAs, 1 MI, 2 PVD) and 2 venous (DVT/PE). Twenty take aspirin, 1 dipyridamole, 1 warfarin and 30 take no thromboprophylaxis. Out of the 24 patients who now have a diagnosis other than IE, 8 have been diagnosed with myelo-proliferative disease. Thirteen patients have a molecular abnormality which is likely to account for their erythrocytosis (11 VHL, 1 PHD-2, 1 EPO-receptor mutations). Three patients have secondary erythrocytosis. Older case studies identified a heterogenous group of patients, some of whom probably had apparent erythrocytosis and some who had either primary polycythaemia or secondary causes later identified (Modan and Modan, Najean et al). More recent reviews have identified a more homogenous group with low rates of transformation to myelofibrosis/acute leukaemia and low rates of thrombosis of around 1% patient-year. Follow up of our initial patient group does indeed reveal a heterogeneous group of patients with 10% now diagnosed with an MPD, although when analysis is confined to those patients who continue to fulfil the criteria for IE, the clinical course has been more stable. There has been no progression to MDS or leukaemia in this group (one patient with PV progressed to AML). The rate of thrombosis is 1.6% patient-years which is lower than the rate seen in PV and is consistent with the rate identified in other series. Molecular defects continue to be identified in this group and future investigation is likely to reveal further abnormalities.