Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Evidence for a direct association with the high conductance calcium-activated potassium channel

Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.     

A comparative investigation of neuroglia in representative vertebrates: a silver carbonate study

The phylogenetic development of neuroglia (astrocytes, oligodendrocytes) was investigated in homologous cortical and subcortical forebrain regions of selected vertebrates. Microglia were not considered in the current study. Four to seven brains from each species were used. Scharenberg's modification for astroglia of del Rio Hortega's silver carbonate technique was used. The analysis of neuroglia cells was based on (1) the characteristic cellular morphology found in each species, (2) a comparison of the selected regions in each animal, (3) the interrelationships of astrocytes and their relations to neurons, blood vessels, and oligodendrocytes. The predominant type of neuroglia found in the fish, frog, and lizard was the ependymal cell; however, non-ependymal glial cells were also present. The bird represented a transitional phylogenetic stage from a predominance of ependymal glial to a predominance of non-ependymal glia. A progressive increase in the morphological relationships of glial cell bodies and processes to neurons was found with ascension of the phylogenetic scale from fish through primate. Interrelations were observed between adjacent astrocytic processes and cell bodies, and between astrocytes and oligodendrocytes. The processes of adjacent glial cells also appeared to show an increase in thickness at the point of approximation. A variety of astrocytes were observed ranging from small, round-oval shaped cells to large polygonal or stellate forms. Variations in the number of astrocytic processes, their thickness, and degree of secondary branching were described, and their possible functional significance was discussed.     

Prospective randomised controlled trial of methods of call and recall for cervical cytology screening.

OBJECTIVE--To discover whether systematic methods of call and recall are more effective than a non-systematic method and to see which of the two systematic methods was more effective. DESIGN--Prospective randomised controlled trial over a year. SETTING--One group general practice. PATIENTS--416 Women over 35 eligible for a smear test who had never had a cervical smear test or in whom a smear test was overdue (previous test more than five years before). INTERVENTIONS--One group received written invitations to have a smear taken. The second group had their notes tagged so that the doctor would remind them (when they attended for another reason) to have a smear test. No special intervention was made in the third group. MAIN OUTCOME MEASURE--Performance of a cervical smear test during the year of the study. RESULTS--32% (45/140) of the screened group, 27% (39/142) of the tagged group, and 15% (20/134) of the control group had a smear test during the year. The percentage of women having a smear test in the screened group was not significantly different from that in the tagged group, but the percentages in the two groups were significantly different from that in the control group. Whether a woman had had a previous smear test significantly affected the uptake of the invitation to have a smear test independently of the method of invitation. CONCLUSIONS--The systematic methods of call and recall were more effective than a non-systematic method. There was no significant difference between the two systematic methods (sending letters or tagging the notes) at one year.     

The effect of phospholipid depletion on the EPR behavior of cytochrome oxidase

Phospholipids are essential components for electron transport activity of cytochrome oxidase. Recently, we have found that the removal of phospholipids from the oxidase affected the copper and low-spin heme signals, and conceivably other paramagnetic centers as demonstrated by EPR spectroscopy. At 4.2–30 °K, the signal amplitudes and power saturation behaviors were studied at approximately g = 2.0 for the copper signal, and in the neighborhood of g = 3.0 for the low-spin heme signal. After depletion of phospholipids the amplitude of the copper signal decreased 25–30% at 12–30 °K and below 12 °K 40–50% under nonsaturating conditions. The amplitude of the low-spin heme signal decreased 60–70% at 4.2–20 °K. Below 14 °K both signals became more resistant to power saturation, but the copper signal was more readily saturated above this temperature, compared to the oxidase with about 25% lipid. After removal of phospholipids, the spectral features of the copper signal remained essentially the same, but the low-spin heme signal broadened and became very asymmetric to show two signals as revealed by the second harmonic EPR spectra. These findings may explain, at least partially, the wide variations in percentage of EPR detectable copper and heme of cytochrome oxidase reported by different laboratories. Unequivocally, the EPR behavior of cytochrome oxidase is not only affected by the protein moiety, but also by the associated phospholipids of the enzyme.     

Bronchial submucosal needle aspiration performed through the fiberoptic bronchoscope

Retrospective review of 251 bronchial submucosal needle aspirations obtained from 171 patients with unsuspected pulmonary malignancy showed that adequate cytologic material was obtained in 87.6% of the aspirations. Eighty-one patients were found to have primary bronchogenic carcinomas (with a visible endobronchial tumor in 39), and 21 had secondary pulmonary metastases. The overall diagnostic accuracy of bronchial submucosal aspiration was 81%, with only four definite false-negative and no false-positive results. The diagnostic accuracy was 82% for bronchial brushing, 71% for forceps biopsy, 67% for postbronchoscopy sputum cytology and 46% for bronchial washing. The major advantage of the submucosal aspiration technique was in evaluating mural mucosal lesions, especially those covered with intact mucosa, that were not readily accessible to forceps biopsy or successful brush sampling. Negative results from a needle aspiration must be interpreted with caution because of the small area a single sample represents. Multiple sequential submucosal aspirations may be helpful in assessing the resectability of bronchial neoplasms. No complications have been encountered with this technique.