Cell biology. Fusion without SNAREs?

Highly orchestrated molecular rearrangements are required for two membranes to fuse, as happens, for example, during neurotransmitter release into the synapse. In an elegant Perspective, Scales et al. discuss two studies (Schoch et al., Wang et al.) that shed new light on the protein interactions involved in membrane fusion.

Paraneoplastic pruritus and alopecia in a horse with diffuse lymphoma.

An 8-year-old castrated male Paint Horse was referred because of generalized pruritus, alopecia, and intermittent fever. Results of gross evaluation of the skin and microscopic evaluation of skin scrapings were inconclusive. Histologic examination of skin biopsy specimens revealed vasculitis. The horse had persistent hypercalcemia; therefore, lymphosarcoma was considered a possibility. Ultrasonography revealed hypoechoic nodules within the parenchyma of the spleen and liver. Results of microscopic evaluation of an aspirate from a splenic nodule were suggestive of lymphosarcoma. The condition of the horse deteriorated, and it was subsequently euthanatized. Postmortem examination revealed diffuse lymphoma. In human beings, generalized pruritus may be an indication of serious internal disease, including malignancy. Paraneoplastic pruritus is most common in patients with Hodgkin's lymphoma but can also develop in patients with non-Hodgkin's lymphoma, leukemia, or a solid neoplasm. Lymphoma should be considered in the differential diagnoses when examining horses with generalized pruritus for which another cause cannot be identified.

Glutamate receptor-mediated calcium entry in neurons derived from P19 embryonal carcinoma cells.

We have examined the control of calcium elevation by glutamate in neurons derived from the mouse P19 embryonal carcinoma cell line. Following transient exposure to retinoic acid, P19 cells differentiate into neurons that express both NMDA and non-NMDA glutamate receptor subtypes. Fluorescence videomicroscopy using the indicator fura-2 revealed concentration-dependent elevation in cytosolic calcium levels with exposure to NMDA or kainate. Replacement of extracellular sodium with N-methylglucamine significantly reduced the action of kainate. Exposure to high K+ medium also elicited an elevation of cytosolic calcium in P19 cells, which was partially inhibited by the calcium channel antagonist nimodipine. These experiments suggest that the elevation in calcium produced by kainate involves the activation of voltage-gated calcium channels as a consequence of membrane depolarization, in contrast to direct calcium entry through NMDA receptor channels. Whole-cell recordings revealed that P19 NMDA receptors were highly permeable to calcium (PCa/PNa = 5.6 +/- 0.2). In most cells, channels gated by kainate displayed low permeability to calcium; the median permeability ratio, PCa/PNa, was 0.053 (range 0.045 to 0.132). Activation of peak currents by NMDA, glycine, and kainate was half-maximal at 24 microM, 240 nM, and 81 microM, respectively. In addition, cadmium-sensitive currents through voltage-gated calcium channels were recorded in P19 cells bathed in barium/TEA chloride. Staining with antibodies directed against AMPA receptor subunits revealed wide-spread immunoreactivity for anti-GluR-B/C and anti-GluR-B/D. About half of the P19 cells were stained with antibodies selective for GluR-D but there was little or no immunoreactivity for the GluR-A subunit.

Synapse formation and establishment of neuronal polarity by P19 embryonic carcinoma cells and embryonic stem cells.

A number of different cell lines that exhibit a partial neuronal phenotype have been identified, but in many cases the full extent of their neuronal differentiation has not been directly addressed by functional studies. We have used electrophysiology and immunofluorescence to examine the formation of synapses and the development of neuronal polarity by murine embryonic stem (ES) cells and the mouse P19 embryonic carcinoma cell line. Within 2-3 weeks after induction by retinoic acid, subsets of P19 and ES cells formed excitatory synapses, mediated by glutamate receptors, or inhibitory synapses, mediated by receptors for GABA or glycine. In ES-cell cultures, both NMDA and non-NMDA receptors contributed to the excitatory postsynaptic response. Staining with antibodies to growth-associated protein-43 and microtubule-associated protein-2 revealed segregation of immunoreactivity into separate axonal and somato-dendritic compartments, respectively. Consistent with our physiological evidence for synapse formation, intense punctate staining was observed with antibodies to the synaptic vesicle proteins synapsin, SV2, and synaptophysin. These results demonstrate the in vitro acquisition by pluri-potent cell lines of neuronal polarity and functional synaptic transmission that is characteristic of CNS neurons.

Time trends and key factors in the choice of one-step or two-step biopsy and surgery for breast cancer.

The relationship between type of biopsy-mastectomy procedure and four sets of independent variables (physician, patient, hospital and tumor characteristics) was examined for 993 locally-staged breast cancer patients diagnosed between 1974 and 1981 in 13 counties of western Washington State. Time trends were also investigated. Cases were selected from records of a population-based cancer registry. The frequency of the two-step procedure, in which biopsy and surgery occur on different days, increased from 28 to 57% during the 8-year study period. Use of the two-step procedure was associated with younger age of the patient, less suspicious symptoms or mammogram results, younger physician cohorts and hospitals with government and health maintenance organization proprietorship. These relationships remained unchanged after controlling for potentially confounding variables. There were also substantial differences between individual hospitals in the frequency of the two-step procedure, suggesting differing schools of thought in different locales.

Uptake and retention of dietary cadmium in mallard ducks.

Adult mallard ducks fed 0, 2, 20, or 200 ppm of cadmium chloride in the diet were sacrificed at 30-day intervals and tissues were analyzed for cadmium. No birds died during the study and body weights did not change. The liver and kidney accumulated the highest levels of cadmium. Tissue residues were significantly correlated in all treatment groups and residues increased with treatment level. Hematocrits and hemoglobin concentrations were normal in all groups throughout the study. Little cadmium accumulated in eggs of laying hens, but egg production was suppressed in the group fed 200 ppm.

Histopathologic effects of dietary cadmium on kidneys and testes of mallard ducks.

Mallard ducks fed 2, 20, or 200 ppm cadmium chloride were sacrificed at 30, 60, and 90 d. No mortality occured during the study and body weights remained unchanged. Kidney weights of the 200-ppm group were significantly greater after 60 and 90 d than those of controls; also, testis weights were significantly lower after 90 d. Kidneys of ducks fed 2 and 20 ppm cadmium were relatively unaffected; however, slight to severe kidney lesions were found in the 200-ppm group after 60 d of treatment. No significant lesions were found in mallard testes after feeding 2 ppm cadmium in the diet, and only a few birds in the 20-ppm group showed slight to moderate gonad alterations. After 90 d of treatment, however, testes of males fed 200 ppm had atrophied and the spermatogenic process had ceased. This study should provide important information for the interpretation of cadmium levels found in kidneys and testes of wild ducks.