The exocytosis of human blood platelets. A fast freezing and freeze-substitution analysis

Human platelets were stimulated with thrombin or collagen in order to induce the release of alpha-granules and dense bodies. The platelets were cryofixed in various stages of exocytosis and subsequently cryosubstituted in acetone containing 4% osmium tetroxide. The platelets embedded in araldite were analyzed in serial sections. The initial changes of the alpha-granules were characterized by an impressive swelling and a dispersal of the granular matrix. Swollen alpha-granules in different stages of exocytosis formed contacts. Between the attached membranes of the alpha-granules electron-dense connections were sometimes observed. In a later stage, the membranes formed a pentalaminar structure (apposition), typical for the prefusion state. After apposition, sequential fusion of single alpha-granules took place and fusions of single or of compound granules with the plasmalemma were observed. The formation of a pore on the platelet surface allowed the passage of granular constituents to the exterior. The dense bodies extruded their electron-dense contents in a similar way after fusion with the plasmalemma but, compared with the alpha-granules, after less extensive swelling. These findings suggest that swelling of the secretory organelles plays an important role for granule fusion and platelet exocytosis. There is some evidence that the characteristic "internal contraction" of cytoskeletal structures in stimulated platelets is not the driving force of the platelet release reaction. An involvement of membranes of the surface connected system in the secretory pathway could not be ascertained.     

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.     

The estrogenic responses to clomiphene in the different cell types of the rat uterus: morphometrical evaluation

The present study was designed to investigate the dose-response of clomiphene on several estrogenic responses in the immature rat uterus and to compare it to available data on estradiol-17 beta. A dissociation was demonstrated among the different estrogenic responses induced by clomiphene. Very high doses of clomiphene were needed to induce the 6-h uterine eosinophilia and deep endometrial edema, and maximal response levels were not reached at any dose studied. On the contrary, many genomic responses were induced with much lower doses of clomiphene, and maximal response levels were reached with at least the two highest doses of clomiphene. This dissociation is in agreement with the existence of separate groups of responses that are mediated by multiple and independent mechanisms of estrogen action involving different kind of receptors. Luminal epithelial, glandular epithelial, and myometrial hypertrophies were also found to differ with regard to the dose needed to induce this response in each cell type. The dissociation between genomic responses of the different uterine cell types supports the hypothesis of different estrogen receptors for each kind of cell. Clomiphene induces mitoses in the different cell types, but the proportion of mitoses in the cell types was different from that described for estradiol. It is suggested that these differences are also due to differences between receptors involved in cell proliferation.     

Thyroid carcinoma: A follow-up study of 11 years

Summary During a follow-up of 11 years of thyroid carcinoma 136 patients were repeatedly examined. 43% papillary, 43% follicular, 11% anaplastic and 2% medullary carcinomas was found. The incidence of these types of carcinoma differed considerably; the frequency peak of papillary carcinomas was reached in 45-year-old humans, that of the follicular carcinomas in people aged 60, that of the anaplastic carcinomas in 70-year-old humans.84% of the patients was female. Classification in pTNM-system: 8% in pT1, 27% in pT2, 12% in pT3 and 49% in pT4. Local and distant metastases were found at a low rate equally in pT1, pT2 and pT3; 26% of patients in pT4 had local metastases and 18% had distant ones in addition. There were 6 patients with metastases of a differentiated adenocarcinoma accumulating no 131-iodine and with no thyroglobulin in serum. 29% of patients had after thyroidectomy an unilateral paresis of the nervus recurrens and 4% a bilateral one. 26% of patients had a permanent hypoparathyroidism after thyroidectomy.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

Microdissection of the Prader-Willi syndrome chromosome region and identification of potential gene sequences

The Prader-Willi syndrome chromosome region on the long arm of human chromosome 15 was microdissected and microcloned from 20 GTG-banded metaphase chromosomes, and 5000 recombinant clones were obtained. Of these clones, 39% identify single-copy human DNA sequences, most of which map to the dissected chromosome region and are evolutionarily conserved in other species. Three of eleven clones studied in detail are deleted in several patients with Prader-Willi syndrome. The microclones will be useful for the physical characterization of the Prader-Willi syndrome chromosome region and the identification of the affected genes in this disease.     

Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells.

Hypervariable minisatellites can be amplified from human DNA by the polymerase chain reaction, using primers from DNA flanking the minisatellite to amplify the entire block of tandem repeat units. Minisatellite alleles up to 5-10 kb long can be faithfully amplified. At least six minisatellite loci can be co-amplified from the same DNA sample and simultaneously detected to provide a reproducible and highly variable DNA fingerprint which can be obtained from nanogram quantities of human DNA. The polymerase chain reaction can also be used to analyse single target minisatellite molecules and single human cells, despite the appearance of spurious PCR products from some hypervariable loci. DNA fingerprinting at the level of one or a few cells therefore appears possible.     

Thermodynamic aspects of plant cell adhesion to polymer surfaces

Summary A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm² which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm²) agree with the pattern of adhesion previously described.