Construction of novel class I histocompatibility antigens by interspecies exon shuffling

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by regulation of hyphal development

The morphological effects of biotin and L-arginine on fruiting body formation of the ascomycete Sordaria macrospora are investigated by scanning electron and light microscopy. Biotin is recognized as an elongation factor and arginine as a branching factor in vegetative and reproductive hyphae. In the absence of exogenous biotin, development is blocked after the ascogonium-core hypha stage of protoperithecial morphogenesis, whereas linear growth of the myceliar front is maintained. The addition of exogenous arginine to a biotin deficient culture induces the formation of numerous side branches even in the older mycelium. Fruiting body formation, however, remains blocked at the protoperithecial stage as before, because of the inability of the side branches to elongate. When biotin and arginine are administered simultaneously, a most vigorous branching and growth are induced in the older mycelium, accompanied by a rapid and maximal formation of fruiting bodies. The results are summarized in a model of the exogenous control of hyphal morphogenesis. The model is designed to explain the relationship between fruiting and hyphal density as well as the edge effect on fruiting body formation.     

Synthesis of linear and comb-like peptide constructs containing up to four copies of a T cell epitope and their capacity to stimulate T cells.

Polypeptide constructs containing up to four copies of the T cell epitope 306-318 of influenza virus haemagglutinin have been synthesized on solid phase. Between the copies, a non-natural PEG-based spacer amino acid has been introduced. The oligomeric epitopes were analysed by RP-HPLC and ES-MS. The arrangement of the epitopes within the peptide constructs was either linear or comb-like. The proliferative response in a T helper cell assay induced by these oligomerized epitopes has been tested, showing that the linearly arranged epitopes are more effective than the comb-like oligomers.     

Characterization of the cryptic Escherichia lineages: rapid identification and prevalence

Strains phenotypically indistinguishable from Escherichia coli and belonging to at least five distinct cryptic lineages, named Escherichia clades I to V, that are genetically divergent from E. coli yet members of the genus have been recently found using multi-locus sequence typing (MLST). Very few epidemiological data are available on these strains as their detection by MLST is not suitable for large-scale studies. In this work, we developed a rapid PCR method based on aes and chuA allele-specific amplifications that assigns a strain a cryptic lineage membership. By screening more than 3500 strains with this approach, we show that the cryptic lineages of Escherichia are unlikely to be detected in human faecal samples (2-3% frequency) and even less likely to be isolated from extra-intestinal body sites (< 1% frequency). They are more abundant in animal faeces ranging from 3-8% in non-human mammals to 8-28% in birds. Overall, the strains from the clade V are the most abundant and from the clade II very rare. These results suggest that members of the cryptic clades are unlikely to be of significance to human and health but may influence the use of 'E. coli' as an indicator of water quality.     

Effects of inhaled corticosteroids on sputum cell counts in stable chronic obstructive pulmonary disease: a systematic review and a meta-analysis

Background

Whether inhaled corticosteroids suppress airway inflammation in chronic obstructive pulmonary disease (COPD) remains controversial. We sought to determine the effects of inhaled corticosteroids on sputum indices of inflammation in stable COPD.

Methods

We searched MEDLINE, EMBASE, CINAHL, and the Cochrane Databases for randomized, controlled clinical trials that used induced sputum to evaluate the effect of inhaled corticosteroids in stable COPD. For each chosen study, we calculated the mean differences in the concentrations of sputum cells before and after treatment in both intervention and control groups. These values were then converted into standardized mean differences to accommodate the differences in patient selection, clinical treatment, and biochemical procedures that were employed across original studies. If significant heterogeneity was present (p < 0.10), then a random effects model was used to pool the original data. In the absence of significant heterogeneity, a fixed effects model was used.

Results

We identified six original studies that met the inclusion criteria (N = 162 participants). In studies with higher cumulative dose (≥ 60 mg) or longer duration of therapy (≥ 6 weeks), inhaled corticosteroids were uniformly effective in reducing the total cell, neutrophil, and lymphocyte counts. In contrast, studies with lower cumulative dose (< 60 mg) or shorter duration of therapy (< 6 weeks) did not demonstrate a favorable effect of inhaled corticosteroids on these sputum indices.

Conclusions

Our study suggests that prolonged therapy with inhaled corticosteroids is effective in reducing airway inflammation in stable COPD.     

Architectural Tradeoffs between Adventitious and Basal Roots for Phosphorus Acquisition

Adventitious rooting contributes to efficient phosphorus acquisition by enhancing topsoil foraging. However, metabolic investment in adventitious roots may retard the development of other root classes such as basal roots, which are also important for phosphorus acquisition. In this study we quantitatively assessed the potential effects of adventitious rooting on basal root growth and whole plant phosphorus acquisition in young bean plants. The geometric simulation model SimRoot was used to dynamically model root systems with varying architecture and C availability growing for 21 days at 3 planting depths in 3 soil types with contrasting nutrient mobility. Simulated root architectures, tradeoffs between adventitious and basal root growth, and phosphorus acquisition were validated with empirical measurements. Phosphorus acquisition and phosphorus acquisition efficiency (defined as mol phosphorus acquired per mol C allocated to roots) were estimated for plants growing in soil in which phosphorus availability was uniform with depth or was greatest in the topsoil, as occurs in most natural soils. Phosphorus acquisition and acquisition efficiency increased with increasing allocation to adventitious roots in stratified soil, due to increased phosphorus depletion of surface soil. In uniform soil, increased adventitious rooting decreased phosphorus acquisition by reducing the growth of lateral roots arising from the tap root and basal roots. The benefit of adventitious roots for phosphorus acquisition was dependent on the specific respiration rate of adventitious roots as well as on whether overall C allocation to root growth was increased, as occurs in plants under phosphorus stress, or was lower, as observed in unstressed plants. In stratified soil, adventitious rooting reduced the growth of tap and basal lateral roots, yet phosphorus acquisition increased by up to 10% when total C allocation to roots was high and adventitious root respiration was similar to that in basal roots. With C allocation to roots decreased by 38%, adventitious roots still increased phosphorus acquisition by 5%. Allocation to adventitious roots enhanced phosphorus acquisition and efficiency as long as the specific respiration of adventitious roots was similar to that of basal roots and less than twice that of tap roots. When adventitious roots were assigned greater specific respiration rates, increased adventitious rooting reduced phosphorus acquisition and efficiency by diverting carbohydrate from other root types. Varying the phosphorus diffusion coefficient to reflect varying mobilities in different soil types had little effect on the value of adventitious rooting for phosphorus acquisition. Adventitious roots benefited plants regardless of basal root growth angle. Seed planting depth only affected phosphorus uptake and efficiency when seed was planted below the high phosphorus surface stratum. Our results confirm the importance of root respiration in nutrient foraging strategies, and demonstrate functional tradeoffs among distinct components of the root system. These results will be useful in developing ideotypes for more nutrient efficient crops.