Two-compartment model for rabbit skin organ culture

Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d. The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid.     

Receptor (CD46) modulation and complement-mediated lysis of uninfected cells after contact with measles virus-infected cells.

Recently, it has been observed that the infection of human target cells with certain measles virus (MV) strains leads to the downregulation of the major MV receptor CD46. Here we report that CD46 downregulation can be rapidly induced in uninfected cells after surface contact with MV particles or MV-infected cells. Receptor modulation is detectable after 30 min of cocultivation of uninfected cells with MV-infected cells and is complete after 2 to 4 h, a time after which newly synthesized MV hemagglutinin (MV-H) cannot be detected in freshly infected target cells. This contact-mediated receptor modulation is also induced by recombinant MV-H expressed by vaccinia virus and is inhibitable with antibodies against CD46 and MV-H. By titrating the effect with MV Edmonston strain-infected cells, a significant contact-mediated CD46 modulation was detectable up to a ratio of 1 infected to 64 uninfected cells. As a result of CD46 downregulation, an increased susceptibility of uninfected cells for complement-mediated lysis was observed. This phenomenon, however, is MV strain dependent, as observed for the downregulation of CD46 after MV infection. These data suggest that in acute measles or following measles vaccination, uninfected cells might also be destroyed by complement after contacting an MV-infected cell.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

Mercury translocation in and evaporation from soil. I. soil lysimeter experiments with 203Hg‐radiolabeled compounds

Due to a considerable increase of anthropogenic mercury emissions, the mercury load of many soils has risen significantly, for instance in northern Europe. Understanding the fate of mercury in soils is a prerequisite for assessing the effects of ecotoxicological concern. This paper presents a method for obtaining qualitative and quantitative information about mercury translocation in and evaporation from soil. Soil lysimeters were treated with ²⁰³Hg‐labeled HgCl₂ and CH₃HgCl and irrigated with artificial rain. It was demonstrated that the leaching of Hg can be detected by measuring the relative y‐activity throughout the soil profile by means of Na(TI)I detectors. Furthermore, the set‐up was designed to allow detection of Hg volatilization from soil by using traps of iodized charcoal, followed by a potassium peroxodisulfate solution and measuring the γ‐activity. The amount of radioactive Hg in soil leachate was measured by a Na(Tl)I well‐type detector after upconcentration. The determination of monomethyl ²⁰³Hg was been performed by extraction procedures that isolate the methyl mercury compounds. The amount of ²⁰³Hg retained in the soil profile and the real depth of leaching were determined by stratifying the soil profile at the end of the experiment and measuring the y‐activity. With control of all pathways of Hg, the experimental design allows performance of a mass balance analysis.     

Characterization of a highly structured domain in Tbp2 from Neisseria meningitidis involved in binding to human transferrin.

The binding of iron-loaded human transferrin at the surface of Neisseria meningitidis is mediated by two polypeptides, Tbp1 and Tbp2. Predicted Tbp amino acid sequences from N. meningitidis strains are highly divergent. This variability is particularly pronounced throughout the Tbp2 polypeptide. In this study, a highly structured and extremely stable Tbp2 domain of about 270 to 290 amino acids which is involved in the binding to transferrin and whose position is well conserved has been characterized. The conservation of such a remarkable structure in a very divergent protein domain (there is only 43% amino acid identity within this region) suggests that is plays an essential biological role and raises a number of questions regarding tbp2 evolution.     

Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

Winter feeding ecology of cod (Gadus morhua) in a fjord of southern Norway

We investigated the winter feeding ecology of cod ( Gadus morhua ) in the Risørfjord and Flødevigen areas on the Skagerrak coast, southern Norway. Diets from the ice-covered Risørfjord were compared with diets from the more coastal Flødevigen area. In the Risørfjord area the diet featured numerically both decapods (56.0%) and fish (27.8%), but fish dominated by mass (75.1%). The most numerous dietary items from the Flødevigen area were decapods (48.1%) and isopods (30.6%), although fish still made up the bulk of the diet's mass (52.6%). Diets at Flødevigen shifted from winter to spring, as polychaetes became important numerically (67.8%) and also contributed substantially by weight (53.6%). Since cod feed opportunistically, differences between areas and seasons probably reflected differences in prey diversity and abundance. Prey size variability increased with increasing predator length, but maximum prey size relative to predator length was constant at about 9%. Prey fish increased in both length and numbers with the length of cod, particularly in the Risørfjord area, where fish were more important in the diet than at Flødevigen. However, the majority of fish consumed in both areas were small gobiids. Based on growth patterns observed in otoliths, with opaque zones (indicating faster growth) formed during winter, the quality and quantity of the winter diet may determine annual growth rates of cod on the Skagerrak coast. Fish and decapods constituted important, high energy food sources, and there was little indication that cod were food limited during winter in this area.