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.     

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.     

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.     

An Ex Vivo Study of Congenital Pigmented Nevi in Epidermal Reconstructs

In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis. All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium. Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure. The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer). Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules. UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis. This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.     

A single chicken anemia virus protein induces apoptosis.

Chicken anemia virus (CAV) causes cytopathogenic effects in chicken thymocytes and cultured transformed mononuclear cells via apoptosis. Early after infection of chicken mononuclear cells, the CAV-encoded protein VP3 exhibits a finely granular distribution within the nucleus. At a later stage after infection, VP3 forms aggregates. At this point, the cell becomes apoptotic and the cellular DNA is fragmented and condensed. By immunogold electron microscopy VP3 was shown to be associated with apoptotic structures. In vitro, expression of VP3 induced apoptosis in chicken lymphoblastoid T cells and myeloid cells, which are susceptible to CAV infection, but not in chicken embryo fibroblasts, which are not susceptible to CAV. Expression of a C-terminally truncated VP3 induced much less pronounced apoptosis in the chicken lymphoblastoid T cells.     

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.     

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.     

OPTIMIZATION OF THE ENUMERATION OF TOTAL AEROBIC BACTERIAL FLORA IN THE FLESH OF SEAFISH

Abstract The total aerobic flora of seafish flesh is weakly halophilic, and requires on average 1.38% NaCl according to statistical studies. Enumeration is optimal on tryptone soya agar or on NaCl supplemented plate count agar (-H₂S), incubated at 20 and 25C, respectively. Plate count agar (-H₂S) was selected because it can also be used for enumeration of hydrogen sulfide-producing bacteria by degradation of sulfur-containing proteins, which are abundant in fish The models employed are sigmoidal. The initial bioburden is too great for there to be a lag phase during storage in ice at 0C. The models show that when the total aerobic microflora count exceeds 100,000 cfu/g, whole or filleted fish stored on ice at 0C are unfit for consumption.