c-Fos-dependent induction of the small ras-related GTPase Rab11a in skin carcinogenesis

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.     

Effect of cyclosporine on the response of normal human lymphocytes to cytomegalovirus in vitro.

Lymphocytes from healthy volunteers seropositive for cytomegalovirus (CMV) demonstrated strong lymphoproliferative responses to CMV-infected and glutaraldehyde-fixed foreskin fibroblasts (CMVFFx) when cocultured for 6 days. Addition of the immunosuppressive drug cyclosporine (CsA) to the cultures resulted in a 10-fold reduction (P less than 0.001) in counts per minute of [3H]thymidine uptake. The proliferative response to noninfected fixed fibroblasts was also reduced 10-fold. Kinetic studies showed an inhibition of the lymphoproliferative response and not an alteration in the time course kinetics in CsA-treated cultures. Cytotoxicity to CMV-infected and unfixed fibroblasts by lymphocytes primed by CMVFFx in the presence of 0.5 micrograms of CsA per ml was significantly reduced (P less than 0.01) as compared with untreated cultures but remained significantly above background level (P less than 0.01). The cytotoxic response was still present but reduced at concentrations of greater than or equal to 1.0 micrograms/ml. Cytotoxicity to noninfected fibroblasts by CMVFFx-primed lymphocytes could be eliminated by as little as 0.5 micrograms of CsA per ml. Stimulation of lymphocytes by CMVFFx but not by noninfected fixed fibroblasts resulted in the in vitro generation of suppressor cells. CsA at a final concentration of 1.0 or 2.5 micrograms/ml did not significantly impair the induction of cells capable of suppressing the lymphoproliferative response of fresh autologous mononuclear cells to CMVFFx. The findings described above may have important clinical implications in that a degree of protective immunity to CMV-infected cells is maintained even in the presence of CsA.     

Protection against murine listeriosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the naturally somatic antigen superoxide dismutase.

A recombinant (r)-Salmonella typhimurium aroA vaccine strain was constructed which secretes the naturally somatic protein of Listeria monocytogenes, superoxide dismutase (SOD), by the HlyB/HlyD/TolC export machinery. Vaccine efficacy of the SOD-bearing carrier strain was compared with that of the p60-secreting construct, S. typhimurium p60s (J. Hess, I. Gentschev, D. Miko, M. Welzel, C. Ladel, W. Goebel, and S. H. E. Kaufmann, Proc. Natl. Acad. Sci. USA 93:1458-1463, 1996). Vaccination of mice with both constructs induced protection against a lethal challenge with the intracellular pathogen, L. monocytogenes. While the somatic listerial antigen, SOD, is immunologically uncharacterized, the naturally secreted protein of L. monocytogenes, p60, is known to be highly immunogenic. Our data emphasize the high vaccine potential of r-Salmonella constructs secreting antigens of somatic or secreted origin. Moreover, they suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated Salmonella spp. as the carrier is capable of potentiating the immune response against foreign proteins independent from their immunogenicity in and display by the natural host.     

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

Characterization of hepatitis B virus (Dane particle)

Hepatitis B virions (Dane particles) were purified from the sera of chronic HBsAg carriers by consecutive rate-zonal and isopycnic centrifugations in sucrose gradients using HBsAg, HBcAg and endogenous DNA polymerase activities as specific markers. Purified Dane particles, radiolabelled with Na ¹²⁵I by the chloramine-T procedure, had a higher buoyant density in CsCl (1.28 g/cm³) than unlabelled particles (1.26 g/cm³) and an estimated sedimentation coefficient of 280 s. ¹²⁵I-Dane particles were fully precipitated by anti-HBs and not by anti-HBc sera. Heavy and light density core particles were purified from heavy and light density populations of Dane particles and radioiodinated. The iodinated polypeptides of Dane particles and HBcAg were compared with those of the iodinated 22-nm form of HBsAg by SDS-PAGE. Iodinated Dane particles contained seven polypeptides with molecular weights of 18,000, 23,000, 26,000, 34,000, 43,000, 48,000 and 115,000. Heavy and light core particles contained three polypeptides with molecular weights of 18,000, 25,000 and 37,000.     

The occurrence, structure and innervation of slow and twitch muscle fibres in the tensor tympani and stapedius of the cat

1. The muscle fibres of the tensor tympani and stapedius of the cat have been examined in the light microscope in teased preparations after cholinesterase staining and in the electron microscope.2. In both muscles, two kinds of fibre have been found: those with an individual end-plate and those with multiple nerve terminals.3. The stapedius fibres with an end-plate have fibrils regularly separated from each other by sarcoplasmic reticulum, a straight Z line, transverse tubular T system elements regularly occurring at the junction of A and I bands, an M line, an extensive sole plate area, and numerous post-junctional sarcolemmal infoldings under the nerve terminal. This type of muscle fibre in the tensor tympani has all of these features except that the fibrils are not well separated from each other, T system elements are absent in some sarcomeres, and a typical M line is absent.4. Compared to the individually innervated fibres, the fibres with multiple endings have fibrils poorly separated from each other by sarcoplasmic reticulum, a jagged Z line, very few T system elements, a less extensive sole plate area, and essentially no folds under the nerve terminal. These fibres in both muscles have M lines.5. Muscle fibres have thus been found in both the tensor tympani and stapedius of the cat which conform in their innervation, the structure of their motor nerve endings, and their internal structure to many of the morphological characteristics which are exhibited by slow muscle fibres elsewhere.     

Comparison between testosterone oenanthate-induced azoospermia and oligozoospermia in a male contraceptive study. IV. Suppression of endogenous testicular and adrenal androgens

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.