Adenovirus serotype determines association and localization of the large E1B tumor antigen with cellular tumor antigen p53 in transformed cells.

The distribution and stability of the cellular tumor antigen p53 were studied in baby rat kidney cells transformed by region E1 sequences of nononcogenic adenovirus (Ad) type 5 (Ad5) or oncogenic type 12 (Ad12). In transformed cells expressing the large E1B T antigen of Ad5, p53 was associated with this T antigen. The complexed proteins were concentrated in a cytoplasmic body, which has been shown to consist of a cluster of 8-nm filaments (A. Zantema et al., Virology 142:44-58, 1985). In transformed cells expressing the E1B region of Ad12, however, no association between the viral large T antigen and p53 was detectable. In the latter case, both proteins were found almost exclusively in the nucleus. The stability of p53 in both Ad5- and Ad12-transformed cells was increased relative to that in primary cells or cells immortalized by the E1A region only. Thus, the increased stability of p53 in Ad-transformed cells is not caused by association with a viral T antigen, but it correlates with expression of E1B and with morphological transformation.     

DETECTION AND ENUMERATION OF H2S+ BACTERIA: APPLICATION TO SHEWANELLA PUTREFACIENS (CIP)

H₂S⁺ bacteria responsible for the degradation of sulfur-containing amino acids of fish muscle are currently little used to evaluate the microbiological pal quality of fish. Shewanella putrefaciens greatly predominates in this flora, and was therefore used to define a suitable culture method and medium. Inoculations by the Spiral surface method at 25C, with an incubation of 72h, gave the best counts on a medium containing two sources of sulfur (organic and inorganic) for H₂S⁺ bacteria. The culture medium and the NaCl concentration were determinant in the evaluation of this flora. At present there is no standard medium which meets these requirements.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Response of a brachytherapy model using 125I in a murine tumor system

The effects of low-dose-rate irradiation (brachytherapy) were investigated in vivo using a murine mammary adenocarcinoma (MTG-B) growing in the flank of C3H mice. For local tumor irradiations, a noninvasive cap was devised to cover the tumor and house three 125I seeds (average apparent activity 5.2 mCi each) located at 120 degree intervals around the circumference of the hemispherical cap (13 mm i.d.). Mice were secured during treatment in a tube allowing limited mobility while restricting access to the seeds. Tumors were exposed to a series of dose rates ranging from 14-40 cGy/h, and the total dose over the treatment interval (48 or 72 h) ranged from 830 to 2378 cGy. A total of nine experiments were conducted using the caps over a 10-week interval. In each experiment three groups (irradiated tumors, sham controls, and untreated controls) were analyzed, each containing 8-15 mice (N = 34, untreated control; N = 46, sham control; N = 91, brachytherapy irradiation). The brachytherapy results are compared to the effects of external beam irradiation in the same tumor system. A linear relationship was observed between the total radiation dose and doubling volume growth delay (GDDV) or treatment volume growth delay (GDTV) for the brachytherapy and external beam irradiation. The slopes of the dose-response curves are steeper for the acute dose (517 cGy/min) external beam irradiation (0.0072 day/cGy, GDDV; 0.00695 day/cGy, GDTV) than for the brachytherapy (0.0050 day/cGy, GDDV; 0.0057 day/cGy, GDTV) using both GDTV and GDDV end points. Comparison of the tumor volume regrowth slopes indicates that the tumor bed effect is larger for external beam irradiation than for brachytherapy, suggesting that the tumor bed effect may be dose-rate dependent.     

Ex Vivo Reconstruction of the Epidermis With Melanocytes and the Influence of UVB

To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm²× 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo.     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

T-cell immunosenescence and inflammatory response in atomic bomb survivors

In this paper we summarize the long-term effects of A-bomb radiation on the T-cell system and discuss the possible involvement of attenuated T-cell immunity in the disease development observed in A-bomb survivors. Our previous observations on such effects include impaired mitogen-dependent proliferation and IL-2 production, decreases in naive T-cell populations, and increased proportions of anergic and functionally weak memory CD4 T-cell subsets. In addition, we recently found a radiation dose-dependent increase in the percentages of CD25(+)/CD127(-) regulatory T cells in the CD4 T-cell population of the survivors. All these effects of radiation on T-cell immunity resemble effects of aging on the immune system, suggesting that ionizing radiation might direct the T-cell system toward a compromised phenotype and thereby might contribute to an enhanced immunosenescence. Furthermore, there are inverse, significant associations between plasma levels of inflammatory cytokines and the relative number of na?ve CD4 T cells, also suggesting that the elevated levels of inflammatory markers found in A-bomb survivors can be ascribed in part to T-cell immunosenescence. We suggest that radiation-induced T-cell immunosenescence may result in activation of inflammatory responses and may be partly involved in the development of aging-associated and inflammation-related diseases frequently observed in A-bomb survivors.