Evidence for the involvement of protein kinase C isoforms in alpha-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells

BACKGROUND: FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of alpha-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2 (PLA2). METHODS: Norepinephrine (NE, 10 mumol/L) was used as an alpha-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo-1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits. RESULTS: Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-alpha, -beta I, -beta II, -gamma, delta, -epsilon, -zeta, and -eta isoforms were identified. Norepinephrine induced translocation of PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon isoforms but not -zeta and -eta from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-alpha. CONCLUSIONS: These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in alpha-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-alpha translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-alpha, to regulate AA generation is an intriguing question that remains to be clarified.     

Correlation between clinical course and quantitative analysis of the ischemia related artery in patients with unstable angina pectoris, refractory to medical treatment. Results of two randomized trials. The European Cooperative Study Group

Patients with unstable angina, refractory to intensive medical therapy, are at high risk for developing thrombotic complications, such as recurrent ischemia, myocardial infarction and coronary occlusion during coronary angioplasty. As both platelet aggregation and/or thrombus formation play an important role in this ongoing ischemic process, a monoclonal platelet GPIIb/IIIa receptor antibody (c7E3) or thrombolytic therapy (alteplase) might be able to modify the clinical course and underlying coronary lesion morphology. To evaluate whether alteplase or c7E3 could influence the incidence of complications, we randomized 36 and 60 patients, respectively to alteplase or placebo, or c7E3 or placebo. All patients exhibited dynamic ECG changes and recurrent pain attacks, despite maximal tolerated medical therapy. Patients were randomized in both studies after initial angiography had demonstrated a culprit lesion amenable for angioplasty. After study drug infusion quantitative angiography was repeated and angioplasty performed. Recurrent ischemia during study drug infusion occurred in 5, 6, 9 and 16 patients from the alteplase, placebo, c7E3 and placebo group, respectively. Major events defined as death, myocardial infarction or urgent intervention occurred in 7, 3, 1 and 7 patients, respectively. Two patients died: one in the alteplase group and one in the placebo group from the c7E3 study. The first patient due to retroperitoneal hemorrhage, the second as a result of recurrent infarction. Qualitative angiography showed resolution of clots in the c7E3 group only, while the same group of patients showed in 20% an improvement in TIMI flow grade, without deterioration in any patient from this group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caudascopic experiences and a new patho-anatomic concept for treatment of sciatica

Osteotomy has been used in treatment of unstable intertrochanteric hip fractures in an attempt to increase the stability of the fracture fragments. We have assessed this stability in a randomised prospective study on 138 patients, all having been fixed by dynamic hip screen, comparing anatomical reduction with medialisation osteotomy. The groups were similar in terms of age, gender and fracture configuration. There was no difference in final results in both groups except that operation time was longer in osteotomy group and the blood loss was more. We found no clear benefit from osteotomy and therefore recommend anatomical reduction and fixation by a sliding hip screw in most cases. Rarely, a fracture configuration which does not allow anatomical reduction may benefit from an osteotomy.     

Net transport of cholesterol from cells of the human EA.hy 926 endothelial cell line to high density lipoproteins

EA.hy 926 cells, a human endothelial cell line, show characteristics of differentiated endothelial cells. The cells express saturable binding of apo E-free 125I-high density lipoprotein3 (HDL3). Bmax increased from 71 to 226 ng HDL3 bound/mg cell protein after cholesterol loading of the confluent endothelial cells with cationized low density lipoprotein (LDL). The affinity did not change after cholesterol enrichment (Kd was 37 micrograms HDL3 protein/ml for control cells and 31 micrograms/ml for loaded cells). Incubation of cholesterol-loaded EA.hy 926 cells with native HDL and LDL had different effects on cellular cholesterol levels. Incubation with HDL decreased both esterified and unesterified cellular cholesterol, but LDL did not change total cellular cholesterol. However, LDL tended to increase cellular cholesteryl esters, with a concomitant decrease of unesterified cellular cholesterol. Incubation of endothelial cells with both HDL and LDL also resulted in decreased total cellular cholesterol levels. These data show that cationized LDL-loaded human endothelial EA.hy 926 cells can be used to study the net transport of cellular cholesterol to HDL, the first step in reverse cholesterol transport.     

Localization of interferon-gamma and Ia-antigen in T cell line-mediated experimental autoimmune encephalomyelitis

This study reports the cellular localization of interferon-gamma (IFN-gamma) and MHC class II antigen (Ia) in the spinal cord of rats with experimental autoimmune encephalomyelitis induced by adoptive transfer of myelin basic protein-specific T cells. Numerous IFN-gamma-positive cells, stained with two different monoclonal antibodies against IFN-gamma, were present from days 3 to 7 after cell transfer. Their number was greatly reduced on day 10. A subpopulation of T cells was IFN-gamma positive. Moreover, a large number of ED1-positive macrophages contained IFN-gamma immunoreactivity. The transient presence of immune cells containing IFN-gamma immunoreactivity in experimental autoimmune encephalomyelitis suggests a pathogenic role of this cytokine in immune-mediated demyelination of the central nervous system.     

Processing attitude statements from in-group and out-group members: Effects of within-group and within-person inconsistencies on reading times.

The authors examined reading times of attitude statements made by group members as a function of consistency of statements with stereotypic expectancies (between-member) and consistency of statements with other statements from the same member (within-member). Stereotype-inconsistent statements were studied longer than consistent statements only when the target group was an outgroup or when subjects were instructed to focus on the group as a whole. Results suggested that the out-group was perceived as a single homogeneous whole regardless of experimental instructions. Inconsistencies within individual group members instigated the longest reading times. This effect was stronger for inconsistencies within out-group members than within in-group members, suggesting that subjects not only expected more within-group variability in in-groups than in out-groups, but they also expected more within-person variability. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A simple and inexpensive method for the identification of Staphylococcus epidermidis and Staphylococcus hominis

Eight hundred and ninety-two strains of Staphylococcus species were identified by means of desferrioxamine susceptibility and fermentation results of three carbohydrates, with the API Staph system (bioMérieux, France) as reference method. No identification could be obtained for 34 strains with API Staph. Of the remaining 858 strains, identical identification was obtained with 842 (98.1%). All 707 strains identified as Staphylococcus epidermidis or Staphylococcus hominis by the API Staph system were found to be desferrioxamine susceptible, and all but 5 (3.3%) of 151 strains identified as other staphylococcal species were found to be resistant, yielding an identification correlation of 99.4% for desferrioxamine. The five additional strains which were susceptible to desferrioxamine were identified as Staphylococcus capitis (2 strains), Staphylococcus lugdunensis (2 strains), and Staphylococcus warneri (1 strain) by API Staph, and as Staphylococcus epidermidis (1 strain), Staphylococcus hominis (3 strains), and one other staphylococcal species by the experimental system.     

Tetracyclines and calcified tissues

Neuronal somata in the rat kidney are very often part of ganglionated plexus and contain nitric oxide synthase (NOS). Examining serial 100 microns slices of whole kidneys, we identified three subpopulations of neuronal somata by: (a) staining for NADPH-diaphorase (NADPH-d) histochemistry followed by the demonstration of dopamine beta-hydroxylase (DBH) by immunoperoxidase, and (b) staining for DBH by immunofluorescence followed by the demonstration of NADPH-d histochemical activity. The largest subpopulation of neuronal somata displayed both DBH immunoreactivity and NADPH-d histochemical activity. The second largest group of somata showed NADPH-d activity only. A small group of neuronal somata showed only DBH immunoreactivity. The presence of catecholaminergic characteristics in NOS-containing neuronal somata is unusual and raises the question as to their origin. Their heterogeneity suggests different functions for the different subpopulations.     

Utility of scintigraphic methods in patients with fever of unknown origin

BACKGROUND: We assessed the utility of scintigraphy with indium 111-labeled polyclonal human IgG scintigraphy in patients with fever of unknown origin that fulfilled the criteria of temperature of 38.3 degrees C or more for at least 3 weeks and no diagnosis during 1 week of hospital admission. We compared the utility of this technique with results of scintigraphic techniques reported in the literature. METHODS: Data for all patients seen at our university hospital in whom 111In-IgG scanning was performed were analyzed and checked for the criteria for fever of unknown origin. The literature on the utility of scintigraphic techniques in patients with fever of unknown origin was reviewed. RESULTS: We studied 24 patients with fever of unknown origin. In 13 patients, focal 111In-IgG accumulation was observed. In nine (38%) of those, the positive 111In-IgG scintigram led to the final diagnosis; in the other four patients (17%), the scintigraphic findings were not helpful. In the 11 patients with negative 111In-IgG scans, extensive diagnostic workup produced no infection as the final diagnosis in nine patients (38%), one had an abscess in a renal cyst that was detected several months later, and in the other the cause of fever was an infected intravenous line. The overall sensitivity and specificity of 111In-IgG scintigraphy were 81% and 69%, respectively. The positive predictive value was 69% and the negative predictive value was 82%. CONCLUSIONS: Our results show that 111In-IgG scintigraphy significantly contributed to the diagnostic process in patients with fever of unknown origin. A positive scan increased the likelihood of finding the cause of the fever, and a negative scan ruled out an inflammatory component with a high degree of certainty. These data compare favorably with data in the literature concerning other radiopharmaceuticals; a larger prospective evaluation of this technique is indicated.