Focal cell contacts detected by ruthenium red, triton X100 and saponin in the granulosa cells of mouse ovary

Granulosa cells in growing follicles of mouse ovary, observed after treatment with ruthenium red (RR) as described by Luft (1971a, b), appeared to be covered by a continuous well-defined layer. On the contrary, treating granulosa cells with 1% Triton X100 (Vaccaro and Brody, 1981), followed by RR staining, resulted in the complete extraction of the plasma membrane coat (Triton does not affect the basement membrane and extracellular matrix proteoglycans). The use of 0.02% saponin together, with the RR stain, or 0.1% Triton X100 followed by RR staining, allows good visualization of follicular basement membrane and extracellular matrix proteoglycans without destroying cell morphology. Using this technique, we observed the extraction of the plasma membrane coat, but focal RR-stained condensations that were unaffected by saponin or 0.1% Triton X100 treatment were observed between plasma membranes of granulosa cells located around the periphery of large Graafian follicles. In some cases, RR condensations were located at the apex of plasmalemmal evaginations, in proximity to adjacent granulosa cells. Focal condensations of RR stain were never observed in secondary follicles. Present evidence suggests that focal cell contacts are mediated by transmembrane intercalated glycoproteins or proteoglycans and consequently play a role in cell adhesion. Their presence among granulosa cells of only very large Graafian follicles may be related to the maturation process of granulosa cells.     

Sequential 1H NMR assignment and secondary structure determination of salmon calcitonin in solution

Salmon calcitonin (sCT) has been investigated by NMR at 500 MHz in a 90% DMSOd6-10% 1H2O (v/v) mixture at 278 K. All backbone and side-chain resonances of the hormone have been assigned by using high-resolution phase-sensitive two-dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects, the NH-alpha CH spin-spin coupling constants, and the 1H/2H exchange kinetics measured in 80% DMSOd6-20% 2H2O (v/v) at 278 K enabled prediction of the secondary structure. Overall, an extended conformation is the dominant feature of the solution, but there are clear indications for a short double-stranded antiparallel beta sheet in the central region comprising residues 12-18, connected by a three-residue hairpin loop formed by residues 14-16. Two tight turns, made by residues 6-9 and 25-28, were also identified, but no evidence was found for the presence of a regular helical segment. The beta sheet favors an amphipathic distribution of the residues, orienting the predominantly hydrophilic Ser13, Glu15, and His17 side chains above the plane of the sheet, and the predominantly hydrophobic Leu12, Gln14, and Leu16 below it. This is interpreted as the "seed" of the amphipathic alpha helix postulated to be responsible for the interaction of sCT with lipids, a situation reminiscent of the folding mechanism of signal peptides in the interaction with membranes. The possible significance of the cis-trans Pro23 isomerism is discussed.     

The influence of the athymic mutation nude on the components of the circadian rhythm of activity in mice

The mutation known as nude brings about the lack of a thymus gland in mice. This immunodeficiency akes it possible to graft normally unaccepted, human cancerous tumors onto the mouse. Consequently, this animal is frequently used as a model for evaluating anti-cancer therapies. The effect of this mutation on biological rhythms constitutes a necessary step before using this model for cancer chronotherapy research. We evaluated the circadian and ultradian components of the rest-activity cycle in the following strains of mice: C57BL/6 with homozygous nu/nu, heterozygous nu/+, thymectomised +/+, and sham-operated +/+. The amount of activity was reduced in nu/nu as compared to the other groups. Nonetheless, neither the nude mutation nor thymectomy yielded any notable change in the circadian rhythm of activity.     

Mathematical analysis of haemopoietic grafted cell renewal and migration through the allogeneic or syngeneic mouse host

A mathematical analysis of results from kinetic studies of 125-iododeoxyuridine uptake and loss in almost all the lymphoid and non-lymphoid organs of mice is described. Applied to data gathered from a graft-versus-host reaction experiment, this analysis affords quantitative precision on the differential effects of organ alloantigens on the proliferating grafted cells. It is shown that, depending on the organ and the post-graft period, cell growth can be ascribed to alloantigen-driven cell renewal or to alloantigen-driven trapping or sequestration. Possible applications of the present approach in graft rejection monitoring are discussed.     

Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-coded tyrosine kinases

The receptors for polypeptide growth factors and proteins coded by oncogenes of the src family are endowed with protein kinase activity and share the uncommon property of autophosphorylating at tyrosine residues. It is unclear whether the tyrosine kinase activity is also directed towards other targets of physiological significance. In this work, phosphotyrosine antibodies were used to detect, by Western blots and immunoprecipitation, proteins phosphorylated at tyrosine in fibroblasts either stimulated by growth factors (PDGF and EGF) or transformed by oncogene-coded tyrosine kinases. In stimulated cells the antibodies detected the autophosphorylated receptors, but only trace amounts of other proteins phosphorylated at tyrosine. In fibroblasts transformed by retroviral oncogenes (v-src, v-abl, v-fps or v-fes) proteins other than the corresponding oncogene-coded kinase, were found. A p70 was found to be heavily phosphorylated in fibroblasts transformed by v-src, v-fes and v-fps. A p130 and a p36 were found in cells transformed by v-src and v-abl. A unique p70 was phosphorylated in v-abl-transformed fibroblasts. These proteins were also phosphorylated in vitro in an immunocomplex kinase reaction. This reaction was blocked by the specific kinase inhibitors. These data strongly suggest that tyrosine kinases phosphorylate protein targets other than themselves. These targets are barely detectable in normal cells stimulated by growth factors, where the kinase activity is triggered rapidly and transiently. By contrast, a number of intracellular proteins phosphorylated at tyrosine accumulate in cells transformed by v-onc-coded kinases, endowed with constitutive and non-regulated enzymatic activity.     

Topological mapping of 13 epitopes on a subunit of Androctonus australis hemocyanin.

A topological localization of epitopes on the surface of the Aa6 subunit of Androctonus australis hemocyanin has been carried out. First, immunocomplex strings composed of native hemocyanin and monoclonal antibodies were examined in the electron microscope and submitted to an image processing by correspondence analysis. The average images were then compared to a three-dimensional model of the 24-mer suggesting that 11 of the 13 epitopes are located in three zones of the subunit surface. Second, the overlaps between the epitopes were then studied by polyacrylamide gel electrophoresis, competitive binding inhibition, and immunoelectron microscopy. Four groups of epitopes were identified. One group was capable of binding exclusively to the free subunit. The other three groups were identical to those found in immunoelectron microscopy. The data are consistent with the existence of a small number of immunodominant regions on the surface of the Aa6 subunit.