Evidence for downregulation of the endothelin-B-receptor by the use of fluorescent endothelin-1 and a fusion protein consisting of the endothelin-B-receptor and the green fluorescent protein.

We generated fusion proteins consisting of the endothelin-B (ET(B))-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ET(B)/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent K(D) values were 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTPgammaS prior to saturation analysis did not alter these values. We also labelled ET-1 with cyanine-dyes (Cy3/ET-1, Cy5/ET-1). In displacement analyses with membranes of MDCK ET(B)/EGFP clones using [125I]ET-1, we found reduced affinity for Cy3/ET-1 and Cy5/ET-1 (about 5- to 10-fold, respectively), but normal efficacy when compared to unlabelled ET-1. Both fluorescent ligands and the ET(B)/EGFP fusion protein were suitable for analysis of receptor trafficking in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ET(B)/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ET(B) receptor and favour its targeting to the lysosomal pathway.

Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor.

A fusion protein consisting of the endothelin B (ET(B)) receptor and the enhanced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ET(B) receptor. The ET(B) receptor and the ET(B)/EGFP fusion protein displayed very similar pharmacological properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temperature PAGE analysis of the (125)I-ET1-bound ET(B) receptor and the (125)I-ET1-bound ET(B)/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ET(B)/EGFP fusion protein demonstrated strong signals at the plasma membrane. On addition of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incubation, ET(B)/EGFP and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using (125)I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ET(B) receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of protease-activated receptors.

The relationship between cognitive ability and positive and negative priming in identity and spatial priming tasks.

The authors examined whether stimulus activation and inhibition in the identity priming task are related to the temporal lobe, and whether these processes in the spatial priming task are related to the parietal lobe. Forty participants performed spatial and identity positive and negative priming tasks, the Vandenberg Mental Rotation task, and the Digit Span task. Both men and women showed significant positive and negative priming in the identity and spatial tasks with no gender difference. The magnitude of identity positive priming was predicted by the Digit Span task, and the magnitude of spatial positive priming was predicted by the mental rotation task. Only women showed a correlation between spatial ability and spatial negative priming. The results are partially consistent with the dorsal-ventral model of cognitive inhibition.

The stability of the circadian rhythm of green finches (Carduelis chloris) under the influence of a weak electrical field.

Free-running activity rhythms of nine green finches (Carduelis chloris) were studied under the influence of a 10-Hz square-wave electrical field. With a field strength of magnitude of E = 2.5 V/m in the empty cage, the population had a mean period of 23.64 +/- 0.77 hr. In the same experiment, but without the electrical field, the period was 23.66 +/- 0.80 hr. These results are in contradiction to Wever's description of a field-induced shortening of the period. A series of experiments with 10-Hz pulses of the same square-wave form, yet with various field strengths (8.7 and 65.2 V/m), also gave no effects.

The prostate of the gorilla.

The prostate gland from a 32-year-old gorilla was examined. The prostate weighed 15 g and was composed primarily of dilated cystic acinar areas with only modest stromal thickening. The acini were lined by a low cuboidal epithelium. A second minor population of smaller glands with extensive papillary projections was also present. The epithelial cells stained densely for acid phosphatase and prostatic-specific antigen. Human prostatic acid phosphatase content of the gland as determined by radioimmunoassay was 2.3 mg/g protein. This is the first published histologic description of a gorilla prostate. The animal was in late middle age but did not display predominant prostatic stromal hyperplasia. Prostatic acid phosphatase from the gorilla cross-reacts immunologically, or is identical to human prostatic acid phosphatase.

Cone-rod dystrophy in the Guinea baboon.

Three stages of macular degeneration associated with diffuse cone-rod dystrophy have been described in a Guinea baboon (P papio) colony. Clinically, the affected animals displayed abnormal behavior associated with decreased vision. Ophthalmoscopically, the lesion in the macula was the only change observable in early cases; retinal vessel attenuation and optic disc pallor were seen only in the advanced cases. The hyperfluorescence of the maculae was the result of loss of pigment in the pigmented epithelium. Electrophysiology supported a cone-rod sequence of this retinal dystrophy. Histologic examination confirmed the theory that the dystrophy began in the cone outer segment but eventually involved all the photoreceptors.