The influence of self-MHC and non-MHC antigens on the selection of an antigen-specific T cell receptor repertoire

We have examined the influence of self-Ag on TCR expression and specificity in the immune response to the Ag pigeon cytochrome c. Previous work has shown that most Ek-restricted cytochrome c-specific T cells from B10 background mice express TCR alpha beta-heterodimers encoded by V beta 3 and V alpha 11 genes, but that T cells expressing V beta 3 proteins are eliminated due to self-tolerance in Mls-2a mouse strains. Thus, EK-restricted cytochrome c-specific T cells from Mls-2a mice fail to express any V beta 3. In the current study the influence of self-MHC and non-MHC Ag on TCR usage in the immune response to cytochrome c was further examined. First, it was demonstrated that the absence of V beta 3 expression in Mls-2a mice does not alter Ir gene function. Specifically, Mls-2a/Eb haplotype V beta 3- [C3H.SW x B10.A(5R)]F1 mice were high responders to cytochrome c despite the fact that previous structure function analyses have shown a very close correlation between Eb-restricted cytochrome c recognition and V beta 3 expression. This demonstration of the plasticity of TCR expression suggests that relatively few Ir gene defects result from tolerance induced by self-Ag. We also examined differences in V alpha 11 expression among cytochrome c-specific T cells from various H-2k haplotype mouse strains. In particular, the low level of expression of V alpha 11 in cytochrome c-specific T cells from C57BR (H-2k) mice was shown not to be due to self-tolerance. Rather, evidence for limited strain polymorphism of V alpha 11 genes, plus the fact that cytochrome c-specific T cells from F1 hybrids between H-2k, Mls-2b identical C57BR and B10.BR mice express high levels of V alpha 11, suggested the possibility that the variable V alpha 11 usage in the cytochrome c-specific responses of these two strains reflected differences in positive selection during ontogeny by non-MHC non-Mls self-Ag.     

Solubilization of covalently bound extensin from capsicum cell walls

Acidified sodium chlorite cleaves isodityrosine and solubilizes covalently bound hydroxyproline-rich material from cell walls. This has been taken as evidence that isodityrosine acts as a cross-link holding the hydroxyproline-rich glycoprotein extensin in the cell wall. However, acidified chlorite was found to cleave peptide bonds in salt-soluble extensin and in bovine serum albumin (BSA). This invalidates the use of conventional acidified chlorite treatment to provide evidence for isodityrosine cross-links. The ratio of BSA:chlorite was important in determining peptidyl cleavage. At a ratio of 0.75:1.00 (mole amino acid residues/mole chlorite), or higher, peptidyl cleavage was not detected. Furthermore, in samples where a low concentration of radioactive extensin was present, BSA substantially protected the peptide bonds of the extensin against peptidyl cleavage during treatment with acidified chlorite, while not preventing the cleavage of isodityrosine. Therefore, acidified sodium chlorite plus BSA was a more specific reagent for the cleavage of isodityrosine than was acidified chlorite alone. This modified treatment solubilized in intact form the `covalently bound' extensin from cell walls of Capsicum frutescens (chili pepper) suspension cultures, providing new evidence compatible with the view that extensin molecules are held in the cell wall by isodityrosine cross-links.     

Microbial Utilization of Estuarine Dissolved Organic Carbon: a Stable Isotope Tracer Approach Tested by Mass Balance

The natural stable isotope values of different plants have been used to trace the fate of organic carbon that enters estuarine ecosystems. Experiments were designed to determine the magnitude of (delta) (sup13)C changes of dissolved organic carbon (DOC) derived from tidal marsh vegetation that occurred during bacterial decomposition. Bacteria were grown on DOC leached from estuarine Spartina alterniflora and Typhus angustifolia plants. In four experiments, 25 to 80% of the initial carbon (2.6 to 9.1 mM organic C) was converted to bacterial biomass and CO(inf2). Mass balance calculations showed good recovery of total C and (sup13)C at the end of these experiments (100% (plusmn) 14% total C; (plusmn) 1(permil) (delta) (sup13)C). The (delta) (sup13)C values of DOC, bacterial biomass, and respired CO(inf2) changed only slightly in the four experiments by average values of -0.6, +1.4, and +0.5(permil), respectively. These changes are small relative to the range of (delta) (sup13)C values represented by different organic carbon sources to estuaries. Thus, microbial (delta) (sup13)C values determined in the field helped to identify the source of the carbon assimilated by bacteria.     

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase.

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 μM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 μM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na⁺/K⁺-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway. © 1996 John Wiley & Sons, Inc.     

Retrotransfer kinetics of R300B by pQKH6, a conjugative plasmid from river epilithon

Abstract The kinetics of conjugation, retrotransfer and mobilisation were studied at 5–15 min intervals between strains of Pseudomonas putida using plasmid pQKH6, isolated from river epilithon, and R300B. Transconjugants from the direct conjugation of pQKH6 and mobilisation of R300B by pQKH6 appeared rapidly, reaching maximum densities within 30–60 min of the start of both filter and liquid mating experiments. However, retrotransconjugants only appeared after a delay. This delay was short (approx. 45–60 min) in filter mating and much longer (2–5 h) for liquid mating experiments. Attempts at predicting the time course of retrotransconjugant development from (1) numbers of transconjugants from the conjugation and mobilisation experiments and (ii) mathematical models based on a mass action approach, both failed to reproduce the observed delay. It was concluded that retrotransfer did not proceed by either a one-step mechanism occuring early in conjugation or two separate conjugation and mobilisation steps. The clear demonstration of a delay in retrotransconjugant formation implies that a new mechanism must be sought. The likely importance of retrotransfer in the environment is discussed.     

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.     

Polygenic Mutation in Drosophila Melanogaster: Estimates from Response to Selection of Inbred Strains

Replicated divergent artificial selection for abdominal and sternopleural bristle number from a highly inbred strain of Drosophila melanogaster resulted in an average divergence after 125 generations of selection of 12.0 abdominal and 8.2 sternopleural bristles from the accumulation of new mutations affecting bristle number. Responses to selection were highly asymmetrical, with greater responses for low abdominal and high sternopleural bristle numbers. Estimates of V(M), the mutational variance arising per generation, based on the infinitesimal model and averaged over the responses to the first 25 generations of selection, were 4.32 X 10(-3) V(E) for abdominal bristle number and 3.66 X 10(-3) V(E) for sternopleural bristle number, where V(E) is the environmental variance. Based on 10 generations of divergent selection within lines from generation 93, V(M) for abdominal bristle number was 6.75 X 10(-3) V(E) and for sternopleural bristle number was 5.31 X 10(-3) V(E). However, estimates of V(M) using the entire 125 generations of response to selection were lower and generally did not fit the infinitesimal model largely because the observed decelerating responses were not compatible with the predicted increasing genetic variance over time. These decelerating responses, periods of response in the opposite direction to artificial selection, and rapid responses to reverse selection all suggest new mutations affecting bristle number on average have deleterious effects on fitness. Commonly observed periods of accelerated responses followed by long periods of stasis suggest a leptokurtic distribution of mutational effects for bristles.     

Intraprotoplasmic feruloylation of arabinoxylans in Festuca arundinacea cell cultures

Graminaceous primary cell walls contain polysaccharides to which are esterified feruloyl residues. Ester biosynthesis is highly specific and the present experiments were performed to ascertain the likely site of feruloylation in living grass cell cultures. Cell cultures of tall fescue grass (Festuca arundinacea Schreber) incorporated exogenous l-[1-³H]arabinose into polymers at a linear rate after a short lag of approx. 1–3 min. Radiolabelled polymers did not start to accumulate in the culture medium until 20–35 min after [³H]arabinose was supplied. However, polymer-bound feruloyl-arabinose residues began to accumulate ³H after a lag of 1–3 min. Assuming that the onset of secretion of radiolabelled polymers into the medium indicates the time before which essentially all the radiolabel was internal to the plasma membrane, the results show that the polysaccharide-bound [³H]arabinose residues must have been feruloylated within the protoplast.Abbreviations AIR alcohol-insoluble residue - BAW butan1-ol/acetic acid/water (12:3:5 by volume) - BEW butan-1-ol/ ethanol/water (20:5:11 by volume) - EPW ethyl acetate/pyridine/ water (8:2:1 by volume) - R_Ara Chromatographic mobility relative to that of l-arabinose We are very grateful to Mr. Gundolf Wende for assistance with the characterisation of the feruloyl esters. K.E.M. is funded by a studentship from the Science and Engineering Research Council in collaboration with Zeneca Agrochemicals.