Monoclonal antibodies to yeast catalase T

Hybridoma cell lines secreting antibodies directed against $\text{Saccharomyces cerevisiae}$ catalase T were constructed by fusing spleen cells of mice immunized with catalase T with P3×63Ag8 mouse myeloma cells. Culture supernatants were assayed for specific antibodies by incubation with ³⁵S-labelled yeast extracts, adsorption of the immune complexes to Protein A — carrying $\text{Staphylococus aureus}$ cells and analysis of the adsorbed yeast proteins by sodium dodecylsulfate gel electrophoresis. Two hybrid clones were isolated mediating adsorption of a protein with electrophoretic mobility of catalase T; one of them, showing considerably higher activity, was characterized further. Anti-bodies produced by this clone belong to the IgG class of immuniglobulins; they can be used for immunoadsorption, but not for direct immunoprecipitation and recognize authentic catalase T as well as catalase T apoprotein.     

A cell based modelling framework for skeletal tissue engineering applications

In this study, a cell based lattice free modelling framework is proposed to study cell aggregate behaviour in bone tissue engineering applications. The model encompasses cell-to-cell and cell–environment interactions such as adhesion, repulsion and drag forces. Oxygen, nutrients, waste products, growth factors and inhibitors are explicitly represented in the model influencing cellular behaviour. Furthermore, a model for cell metabolism is incorporated representing the basic enzymic reactions of glycolysis and the Krebs cycle. Various types of cell death such as necrosis, apoptosis and anoikis are implemented. Finally, an explicit model of the cell cycle controls the proliferation process, taking into account the presence or absence of various metabolites, sufficient space and mechanical stress. Several examples are presented demonstrating the potential of the modelling framework. The behaviour of a synchronised cell aggregate under ideal circumstances is simulated, clearly showing the different stages of the cell cycle and the resulting growth of the aggregate. Also the difference in aggregate development under ideal (normoxic) and hypoxic conditions is simulated, showing hypoxia induced necrosis mainly in the centre of the aggregate grown under hypoxic conditions. The next step in this research will be the application of this modelling framework to specific experimental set-ups for bone tissue engineering applications.     

Immunology of Staphylococcal biofilm infections in the eye: new tools to study biofilm endophthalmitis

Endophthalmitis is an important disease of the eye that is most frequently caused by postoperative and post-traumatic introduction of bacteria into the posterior segment of the eye. In the case of severe infections, visual acuity is greatly damaged or completely lost. Much work has focused on the ability of planktonic bacteria to cause infection and ocular damage while little work has focused on chronic infections in endophthalmitis mediated by the formation of bacterial biofilms on the surface of the lens. This review focuses on the interaction of Staphylococcus aureus and Staphylococcus epidermidis lens-associated biofilms in endophthalmitis. Additionally, this review highlights some relevant biofilm-immune system interactions and outlines a new in vivo mouse model to explore biofilm-related infections in endophthalmitis.     

The Pmed1 gene promoter of human Fc gamma RIIIA can function as a NK/T cell-specific restriction element, which involves binding of Sp1 transcription factor

The low-affinity receptor for IgG (human FcgammaRIIIA) is selectively expressed by a subset of T lymphocytes, NK cells, and macrophages. To understand the mechanisms underlying this pattern of cell type-specific expression, we initially identified alternative promoters, Pmed1/2 and Pprox, in the 5' end of the FcgammaRIIIA gene. In this study, we focused on the Pmed1 promoter and demonstrated this 93-bp region to be highly specific in governing restriction to NK/T cell lines. This property of Pmed1 is context independent and can extend to a disparate promoter. Deletion analysis defined a contribution of two separate elements located to the 5' 21-bp (-942/-922) and 3' 72-bp (-921/-850) regions of Pmed1 in conferring NK/T cell specificity. The 5' part of Pmed1 contains binding sites for Sp1 and NK element-recognizing factors and substitution mapping studies revealed a critical requirement of the Sp1-I site. The importance of Sp1 protein to regulate maximal Pmed1 promoter activity was further established by EMSAs and cotransfection experiments in Sp1-null Drosophila SL2 cells. Our data suggest that Sp1 can contribute, in part, to NK/T cell restriction and further indicate that the FcgammaRIIIA Pmed1 sequence might be useful to direct the NK/T cell-specific expression of heterologous genes.     

Are rocky shore ecosystems affected by nutrient-enriched seawater? Some preliminary results from a mesocosm experiment

The response of rocky shore ecosystems to increased nutrient availability was examined in eight land-based mesocosms designed for hard-bottom littoral communities built at Marine Research Station Solbergstrand (Norway). The average seawater volume in each basin was 9 m³ with an average water residence time of about 2 h. A tidal regime resembling that in the fjord was maintained in the basins, and waves were generated regularly. NH₄NO₃ and H₃PO₄, at a constant molar NP ratio of 16:1, was added into 6 basins at concentrations 1, 2, 4, 8, 16, 32 M DIN above the background DIN concentration during 1 1/2 years. Two mesocosms were kept as control treatment. Marine communities were introduced into the basins two years prior to the start of nutrient dosage. The effects of nutrient enrichment were few and only marginal during the first year of nutrient addition, while some effects became more obvious during the second year. The growth rate of the periphyton and fast-growing macroalgae communities was stimulated by nutrient enrichment, while the response was less evident among the perennial fucoids. The structure of the macroalgal communities, however, did not change during 16 months' measurements. In contrast, growth on artificial rock substrates during the same period of time revealed intensive growth of the fast-growing Ulva lactuca in high-dosed basins compared with low-dosed and control basins, which were dominated by the fucoid Fucus serratus. The fauna communities exhibited only a minor response to nutrient treatment. The common periwinkle Littorina littorea, however, appeared with increased abundance in the high-dosed basins. The total system metabolism tended to increase slightly, but not significantly, with increased nutrient loading.     

Opposite regulation of type II and III receptors for immunoglobulin G in mouse glomerular mesangial cells and in the induction of anti-glomerular basement membrane (GBM) nephritis

We examined the capacity of mouse glomerular mesangial cells (MC) to express and function through two different low affinity FcgammaRs, the activating FcgammaRIII and the inhibitory FcgammaRII. Immunohistochemistry identified FcgammaRII as the prominent FcgammaR in the kidney, and low levels of FcgammaRIIb2-specific mRNA were also detected in primary cultures of growth-arrested MC. Activation by tumor necrosis factor-alpha/interleukin-1beta induced substantial FcgammaRII expression in proliferating MC. Importantly, however, stimulation with interferon-gamma (IFN-gamma)/lipopolysaccharide or IFN-gamma alone resulted in a complete down-regulation of FcgammaRII, which was accompanied by a strong increase in FcRgamma chain mRNA and a surface appearance of FcgammaRIII. Activating FcgammaRIII triggered mRNA synthesis for monocyte chemoattractant protein-1 (MCP-1), MCP-5, cytokine-induced neutrophil chemoattractant, and RANTES, whereas FcgammaRIII-deficient MC failed to respond to immune complex (IC) activation as shown by impaired production of MCP-1 mRNA/protein. In a passive model of acute anti-glomerular basement membrane (GBM) nephritis, induction of FcgammaRIII and suppression of FcgammaRII occurred in kidney tissues. Blockade of FcgammaRII, when induced selectively in the kidney, resulted in enhanced inflammation. Taken together, our results define a novel regulatory pathway with opposite regulation of FcgammaRII (suppressed) and FcgammaRIII (induced) by IFN-gamma on MCs in vitro and anti-GBM IgG in vivo. Herein is provided the first evidence that glomerular FcgammaRII plays an important immunoregulatory role in the initiation of IC glomerulonephritis.     

Heterogeneity and Photoinhibition of Photosystem II Studied with Thermoluminescence

Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone Q_B⁻ with the S₂ state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S₂Q_A⁻ recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50°C in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a Tyr_D⁺-Q_A⁻ recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42°C. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52°C) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45°C (C-band) slightly increased. It is proposed that the 42 to 52°C band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII Q_B-nonreducing centers.     

The influence of metal cations and pH on the heat sensitivity of photosynthetic oxygen evolution and chlorophyll fluorescence in spinach chloroplasts

The heat-sensitivity of photosynthetic oxygen evolution of thylakoids isolated from spinach increases by increasing the pH above neutral value. The temperature for inactivation (transition temperature) is lowered from about 45° C (pH 6.0–7.4) to 33°C (pH 8.5). Similar results are obtained with intact chloroplasts. At pH 7.0 the transition temperature of washed thylakoids decreases by lowering the salt concentration below 20 mM with monovalent cations (Li⁺, Na⁺, K⁺) and below 3–4 mM with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺). Illumination decreases the heat-sensitivity of oxygen evolution in intact chloroplasts, but even increases the heat-sensitivity in uncoupled chloroplasts. In intact chloroplasts the transition temperature of the heat-induced rise in chlorophyll fluorescence yield (F_o; see Schreiber and Armond 1978) decreases from 44° C to 38° C when the pH of the suspending medium is increased from 6.5 to 8.5. At 20° C, F_o is almost insensitive to pH (6.0–8.5). At 40° C, however, F_o is constant between 6.0 and 7.0, but strongly increases by increasing the pH above neutral value. The results are discussed in terms of a close relation between electrostatic forces at the thylakoid membrane and thermal sensitivity of photosynthetic apparatus. It is suggested that the heat-sensitivity of the photosystem II complex partially depends on the ionization state of fixed groups having alkaline pK. The packed volume of thylakoids suspended in a low salt medium increases when the temperature is increased above 30° C (pH 7.0) and above 20° C (pH 8.0), respectively. This result suggests a heat-induced increase in surface charge density of the thylakoid membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES morpholinoethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - TRICIN N-[tris(hydroxymethyl)-methyl] glycine     

Das Haustorium von Cassytha pubescens R. Br.

Ohne ZusammenfassungMit 19 Textabbildungen.