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.     

Cellular roles of kinesin and related proteins

Frequency response characteristics of six popular stethoscopes are reported for the higher frequency range (to 3000 Hz) to supplement equivalent measurements for the lower frequencies (35-1000 Hz) published previously. Spectra of the sounds of swallowing from the throat, transduced with an accelerometer, demonstrate important frequency composition in this higher range. Two stethoscope models were found to have superior transmission characteristics for use in cervical auscultation of swallowing sounds.     

Respective importance of the different risk factors for osteoporosis

Among the factors which influence accumulation of bone mass, genetic factors related to race and family are preponderant: they account for approximately 80% of the variance observed in adulthood. At puberty, nutritional factors and physical activity modulate the accumulation of bone and determine whether or not optimal bone mass is achieved. In women, estrogen deprivation following menopause, and also more minor disorders of gonad function, are the main causes of decreased bone mass; their respective contributions are analyzed.     

A genomic map of infectious laryngotracheitis virus and the sequence and organization of genes present in the unique short and flanking regions

The involvement of the intermediate area and B?tzinger complex (BOT) of the rostral ventral respiratory group (r-VRG) in laryngeal control and generation of the expiration reflex were studied in anaesthetized non-paralyzed cats Focal cooling (to 20 degrees C) of the nucleus paraambigualis (NPA) caused changes in the frequency and timing of breathing with the concomitant rise in laryngeal resistance. Cooling of the nucleus ambiguus resulted in a consistent drop in laryngeal resistance. Alterations in timing and intensity of breathing but no changes in laryngeal patency were found during cooling of the BOT. The expiration reflex was inhibited by cooling of either the NPA or BOT. The role of these medullary regions in the control of laryngeal patency and central integration of the expiration reflex is discussed.     

Quantitative methods for scoring cell migration and invasion in filter-based assays

The ability of cells to traverse pores in a biocompatible filter provides means for examining cell chemoattraction. Filter-based assays also permit rapid, quantitative assessment of the in vitro migratory and invasive potential of tumor cells. Scoring migration has relied on visual counting of stained cells which appear on the underside of the filter and determining a true percentage score involves arduous counting of cells on both filter surfaces. Visual counting of random fields may be unreliable, and counting all fields is laborious. In the present study we developed and compared two alternative methods for scoring cell numbers in filter-based assays, a colorimetric assay of toluidine blue binding, and a radioassay of cells prelabeled with [3H]thymidine. Each method was evaluated for sensitivity, variability, ease of use and efficiency, and suitability for use in assays of cell migration and invasion. The radiolabeling method proved to be sensitive and reliable and was the most efficient technique. Although less sensitive and specific, the colorimetric dye method offered a rapid and reliable, nonradioactive alternative with the distinct advantage of preserving intact cultures for follow-up visual assessments. We conclude that colorimetric and radiolabel scoring of filter-based assays are reliable and efficient semiautomated methods which provide means to obtain more complete assessments of cell migration and invasion.     

Lung function measurement in general practice: a comparison of the Escort spirometer with the Micromed turbine spirometer and the mini-Wright peak flow meter

It is important that new types of spirometer for widespread clinical use are pragmatically evaluated in primary care. This study compared measurements taken by a new portable Fleisch pneumotachograph spirometer (known as the Escort) with those of the commonly used mini-Wright peak flow meter and the Micromed Pocket turbine spirometer. A pragmatic study was conducted in two phases during routine surgeries at Aldermoor Health Centre, Southampton. Phase I compared the new spirometer with the mini-Wright peak flow meter and Phase 2 compared the new spirometer and the turbine spirometer. One hundred patients aged 5-88 years (56 patients with a history of chronic respiratory complaints and 44 patients without) entered Phase 1, and 100 patients aged 6-82 years (62 patients with a history of chronic respiratory complaints and 38 patients without) entered Phase 2. Each patient contributed only once to each phase, but some entered both phases on separate occasions. Ninety-five percent limits of agreement (mean +/- SD) were wide for all comparisons. Graphical plots revealed trends towards higher Escort values as mean values rose compared with both mini-Wright and turbine readings for peak expiratory flow rate and forced expiratory volume in one second. Possible over-reading of peak expiratory flow rate with the mini-Wright meter at low mean values was also seen. Readings taken with these different types of meter cannot be interchanged with confidence in clinical practice. The clinical significance of the theoretically more accurate measures of lung function produced with the new meter, and indeed of spirometry itself, needs further investigation.     

Simple method for determination of dietary fiber in frozen foods

A simple method was developed for the determination of dietary fiber in multicomponent foods. The method involves dispersing the sample into pH 7.4 phosphate buffer and adding bile and pancreatic enzyme as described. Results were comparable to AOAC methods with correlation coefficients of 86% for multicomponent dinners and 89% for breakfast foods. Coefficients of variation ranged from 7.4 to 20.0% for multicomponent foods and 1.0 to 3.6% for single component foods. In addition, blind duplicate samples had a correlation of 0.99. The described method required less time, labor, and manipulation than AOAC methods.