Glucocorticoid regulation of calcium-activated potassium channels mediated by serine/threonine protein phosphatase

Adrenal glucocorticoids exert powerful effects on cellular excitability in neuroendocrine cells and neurons, although the underlying mechanisms are poorly understood. In metabolically intact mouse anterior pituitary corticotrope (AtT20) cells glucocorticoid-induced proteins render large conductance calcium-activated potassium (BK) channels insensitive to inhibition by protein kinase A (PKA). In this study we have addressed whether this action of glucocorticoids is mediated via protein phosphatase activity at the level of single BK channels. In isolated inside-out patches from control AtT20 cells BK channels (125 pS) were inhibited by activation of closely associated PKA. Pretreatment (2 h) of cells with 1 microM dexamethasone before patch excision did not modify the intrinsic properties or expression levels of BK channel alpha-subunits in AtT20 cells. However, PKA-mediated inhibition of BK channel activity in isolated patches from steroid-treated cells was severely blunted. This effect of steroid was not observed using adenosine 5'-O-(3-thiotriphosphate) as phosphate donor or on exposure of the intracellular face of the patch with 10 nM of the protein phosphatase inhibitors okadaic acid or calyculin A but was mimicked by application of protein phosphatase 2A (PP2A) to the intracellular face of patches from control cells. Glucocorticoids did not modify total PP2A activity in AtT20 cells, suggesting that modified PP2A-like phosphatase activity closely associated with BK channels is required for glucocorticoid action.     

Survey of incidence of Clostridium difficile infection in Canadian hospitals and diagnostic approaches

A questionnaire relating to Clostridium difficile disease incidence and diagnostic practices was sent to 380 Canadian hospitals (all with > 50 beds). The national questionnaire response rate was 63%. In-house testing was performed in 17.6, 61.5, and 74.2% of the hospitals with < 300, 300 to 500, and > 500 beds, respectively. The average test positivity rates were 17.2, 15.3, and 13.2% for hospitals with < 300, 300 to 500, and > 500 beds, respectively. The average disease incidences were 23.5, 30.8, and 40.3 cases per 100,000 patient days in the hospitals with < 300, 300 to 500, and > 500 beds, respectively. In the 81 hospitals where in-house testing was performed, cytotoxin testing utilizing tissue culture was most common (44.4%), followed by enzyme-linked immunosorbent assay (38.3%), culture for toxigenic C. difficile (32.1%), and latex agglutination (13.6%). The clinical criteria for C. difficile testing were variable, with 85% of hospitals indicating that a test was done automatically if ordered by a doctor. Our results show that C. difficile-associated diarrhea is a major problem in hospitals with > or = 200 beds. Despite a lower disease incidence in smaller hospitals, there was a higher diagnostic test positivity rate. This may reflect the preference of smaller hospitals for culture and latex agglutination tests.     

Downregulation of BDNF mRNA and protein in the rat hippocampus by corticosterone

Previously, we showed that corticosterone regulates BDNF mRNA levels in the hippocampus. In the present study, we have investigated the time course and dose-dependency of this effect at both the mRNA and the protein level. Corticosterone was administered in doses of 30 and 1000 microgram/kg b.w. subcutaneously to adrenalectomized animals. At 3, 6, 12 and 24 h after administration BDNF and trkB mRNA levels in hippocampal subfields were measured by in situ hybridization. Our results show a dose-dependent decrease in BDNF mRNA in dentate gyrus and CA1 at 3 h. After the high dose, this decrease was 70% and 40% respectively. In addition, ELISA was performed to study if this downregulation is also detectable at the protein level. Hippocampal tissue was used from adrenalectomized animals which had received 1000 microgram/kg b.w. corticosterone 4 and 6 h before decapitation. At both time points, a decrease in BDNF protein was observed; 17% at 4 h and 14% at 6 h after corticosterone, as compared to the vehicle injected controls. TrkB mRNA levels were not affected by corticosterone. However, between 6 and 24 h after treatment, increases in trkB mRNA were observed. In conclusion, we have found a transient, dose-dependent decrease in BDNF mRNA and protein in the hippocampus, which may underly changes in neuronal plasticity in the hippocampus after short-term changes in corticosterone concentrations.     

Changes of endothelin-1 and atrial natriuretic peptide during dobutamine stress echocardiography

The aim of this study was to test the hypothesis that plasma endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) concentrations in patients with ischemic heart disease are related either to myocardial ischemia or left ventricular (LV) dysfunction during dobutamine stress echocardiography. Plasma concentrations of ET-1 and ANP were measured in three patient groups. Group I (n = 21) patients had normal stress echocardiography and a resting LV ejection fraction (LVEF) of 40% or more. Group II (n = 32) had positive stress echocardiography and a resting LVEF of more than 40%. Group III (n = 18) had positive stress echocardiography with a resting LVEF of less than 40%. All three groups were subjected to thallium 201 scintigraphy and coronary angiography studies. The resting LV end-diastolic pressure was significantly higher in groups II and III than in Group I. The LVEF decreased significantly in group III compared to groups I and II. In the resting state, groups II and III had higher ET-1 concentrations than Group I (p = 0.021 and p = 0.039, respectively). The plasma ANP concentration was higher in group III than in groups I and II (p = 0.005 and p = 0.054, respectively). During peak dobutamine infusion, the ET-1 concentration dropped 8.7% from the baseline in group I, 10.2% in group II, and 10.5% in group III. The ANP concentrations were increased in all three groups but only the increase in Group II reached statistical significance. In conclusion, in patients with suspected ischemic heart disease, the concentrations of ET-1 and ANP may predict significant anatomic and functional coronary artery disease. However, ET-1 does not play a pathophysiologic role during an ischemic attack.     

Oral chemotherapy: rationale and future directions

PURPOSE AND METHODS: The expanding role of oral chemotherapy in oncology is suggested by the abundance of orally formulated agents currently in development. The pharmacoeconomic principles that drive oral drug formulation are discussed. Patient preference for oral therapy is identified as a second major impetus for the design of oral cytotoxics. While the rationale for oral formulations is apparent, substantial patient compliance and pharmacokinetic limitations have been identified for this route of administration. Specific aspects of bioavailability limitations and patient compliance are discussed. Relevant pharmacokinetic data for each orally formulated chemotherapy agent are compared and selected novel oral cytotoxics and cytotoxic modulators are discussed. RESULTS: A review of pharmacokinetic literature suggests substantial variability in bioavailability for many orally formulated cancer cytotoxics. While these findings are observed for all classes of oral drugs, the issue is especially critical for cancer chemotherapy, in which a narrow therapeutic index is frequently observed. Improved bioavailability and reduced interpatient biovariability are therefore desirable for new cytotoxic formulations. Pharmacologic manipulations to improve bioavailability and reduce costs are examined. CONCLUSION: Oral chemotherapy represents a fundamental change in contemporary oncology practice, driven by pharmacoeconomic issues, patient convenience, and the potential for improved patient quality of life. Novel cytostatic therapies that require protracted drug administration periods will also favor an oral formulation. While the use of oral chemotherapy may initially be limited to metastatic disease palliation, demonstration of equivalent efficacy would allow for its subsequent use in adjuvant settings. This efficacy is contingent on circumventing bioavailability limitations and patient noncompliance. The development of specific, low-toxicity inhibitors of CYP3A4, P-glycoprotein (P-gp), and other drug metabolizing enzymes such as dihydropyrimidine dehydrogenase represents a major innovative step in the successful formulation of oral chemotherapy.     

Primary testicular and paratesticular tumors of childhood

Testicular and paratesticular neoplasms are uncommon tumors of childhood. Consequently, the experience gained with regard to their optimal management is limited in any given children's cancer centre. Here we review the classification, diagnosis, and staging of testicular and paratesticular neoplasms and subsequently discuss the more frequently occurring ones: germ cell tumors, gonadal stromal tumors, gonadoblastoma, tumors of the supporting tissue, lymphomas and leukemias, tumor-like lesions, secondary tumors, and tumors of the adnexa.     

The role of PET-FDG in questionable diagnosis of relapse in the presence of radionecrosis of brain tumors

INTRODUCTION: Although CT and MR are sensitive techniques for the detection of cerebral tumours, both have limitations in distinguishing between tumour relapse (TR) and post-treatment radionecrosis (RN). PATIENTS AND METHODS: In this study we have determined the usefulness of metabolic imaging with PET-FDG in such situations. We assessed 70 patients with CNS tumours (22 low grade astrocytomas, 25 high grade astrocytomas, 3 oligodendrogliomas, 13 metastatic tumours and 7 other tumours. All had been treated with radiotherapy and other treatments such as radiosurgery, chemotherapy or different types of surgery, and presented clinical pictures which made it necessary to decide the differential diagnosis of relapse or radionecrosis. RESULTS: In the PET-FDG study visual and semiquantitative analysis was done by SUV (Standardized Update Value). Confirmation of the findings was obtained in 44 cases (24 TR and 20 RN). MR was doubtful or inconclusive in most cases, whilst with PET correct diagnosis was made in all cases. CONCLUSIONS: Metabolic imaging with PET-FGD is better than anatomostructural imaging techniques for differential diagnosis between tumour relapse and radionecrosis in CNS tumours which have been treated. Prospective studies are necessary for evaluation of SUV as a factor for prognosis of survival.     

Identification of sorting determinants in the C-terminal cytoplasmic tails of the gamma-aminobutyric acid transporters GAT-2 and GAT-3

In order to perform their physiologic functions, polarized epithelial cells must target ion transport proteins to the appropriate domains of their plasma membranes. Molecular signals responsible for polarized sorting have been identified for several membrane proteins which span the bilayer once. Most ion transport proteins are polytopic, however, and little is known of the signals responsible for the targeting of this class of polypeptides. Members of the gamma-aminobutyric acid (GABA) transporter family are polytopic membrane proteins found endogenously in both epithelial cells and neurons. We have identified narrowly defined sequences which are required for the proper accumulation of two members of this transporter family in Madin-Darby canine kidney cells. The highly homologous GABA transporter isoforms, GAT-2 and GAT-3, localize to the basolateral and apical surfaces, respectively, when expressed stably in Madin-Darby canine kidney cells. We have generated deletion constructs and chimeric transporters composed of complimentary portions of GAT-2 and GAT-3. We find that information which directs their differential sorting is present in the C-terminal cytoplasmic tails of these two polypeptides. A sequence of 22 amino acids at the C terminus of GAT-2 is required for the transporter's basolateral distribution and is capable of directing GAT-3 to the basolateral surface when appended to the C terminus of this normally apical polypeptide. The deletion of 32 amino acids from the C terminus of GAT-3 causes this transporter to become mislocalized to both surfaces. Moreover, removal of the final three amino acids of GAT-3 (THF) similarly disrupts its apical sorting. The GAT-3 C-terminal sequence resembles motifs which interact with PDZ domains, raising the possibility that the steady state distribution of GAT-3 at the apical plasmalemmal surface requires a protein-protein interaction mediated by its extreme C-terminal cytoplasmic tail. These data provide the first characterization of a protein-based signal required for the apical distribution of a membrane protein.