Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding

A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.     

Suppression of the biocontrol agent trichoderma harzianum by mycelium of the arbuscular mycorrhizal fungus glomus intraradices in root-free soil

Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne plant pathogens. However, possible adverse effects of this fungus on arbuscular mycorrhizal fungi might be a drawback in its use in plant protection. The objective of the present work was to examine the interaction between Glomus intraradices and T. harzianum in soil. The use of a compartmented growth system with root-free soil compartments enabled us to study fungal interactions without the interfering effects of roots. Growth of the fungi was monitored by measuring hyphal length and population densities, while specific fatty acid signatures were used as indicators of living fungal biomass. Hyphal 33P transport and beta-glucuronidase (GUS) activity were used to monitor activity of G. intraradices and a GUS-transformed strain of T. harzianum, respectively. As growth and metabolism of T. harzianum are requirements for antagonism, the impact of wheat bran, added as an organic nutrient source for T. harzianum, was investigated. The presence of T. harzianum in root-free soil reduced root colonization by G. intraradices. The external hyphal length density of G. intraradices was reduced by the presence of T. harzianum in combination with wheat bran, but the living hyphal biomass, measured as the content of a membrane fatty acid, was not reduced. Hyphal 33P transport by G. intraradices also was not affected by T. harzianum. This suggests that T. harzianum exploited the dead mycelium but not the living biomass of G. intraradices. The presence of external mycelium of G. intraradices suppressed T. harzianum population development and GUS activity. Stimulation of the hyphal biomass of G. intraradices by organic amendment suggests that nutrient competition is a likely means of interaction. In conclusion, it seemed that growth of and phosphorus uptake by the external mycelium of G. intraradices were not affected by the antagonistic fungus T. harzianum; in contrast, T. harzianum was adversely affected by G. intraradices.     

In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.     

Caspase-1 and caspase-8 cleave and inactivate cellular parkin

Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons, thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage, and the major cleavage site is after Asp126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK-293 cells identified caspase-1, -3, and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9, and -11 did not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8, but not by caspase-3. In anti-Fas-treated Jurkat T cells, parkin cleavage was inhibited by caspase inhibitors hFlip and CrmA (but not by X-linked inhibitor of apoptosis (XIAP)), indicating that caspase-8 (but not caspase-3) is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3-deficient MCF7 cells, either by caspase-1 or -8 overexpression or by tumor necrosis factor-alpha treatment, led to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin and suggest that death receptor activation and inflammatory stress can cause loss of the ubiquitin ligase activity of parkin, thus causing accumulation of toxic parkin substrates and triggering dopaminergic cell death.     

No significant contribution of arbuscular mycorrhizal fungi to transfer of radiocesium from soil to plants

The diffuse pollution by fission and activation products following nuclear accidents and weapons testing is of major public concern. Among the nuclides that pose a serious risk if they enter the human food chain are the cesium isotopes 137Cs and 134Cs (with half-lives of 30 and 2 years, respectively). The biogeochemical cycling of these isotopes in forest ecosystems is strongly affected by their preferential absorption in a range of ectomycorrhiza-forming basidiomycetes. An even more widely distributed group of symbiotic fungi are the arbuscular mycorrhizal fungi, which colonize most herbaceous plants, including many agricultural crops. These fungi are known to be more efficient than ectomycorrhizas in transporting mineral elements from soil to plants. Their role in the biogeochemical cycling of Cs is poorly known, in spite of the consequences that fungal Cs transport may have for transfer of Cs into the human food chain. This report presents the first data on transport of Cs by these fungi by use of radiotracers and compartmented growth systems where uptake by roots and mycorrhizal hyphae is distinguished. Independent experiments in three laboratories that used different combinations of fungi and host plants all demonstrated that these fungi do not contribute significantly to plant uptake of Cs. The implications of these findings for the bioavailability of radiocesium in different terrestrial ecosystems are discussed.     

High avidity antigen-specific CTL identified by CD8-independent tetramer staining

Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.     

Volatile compounds produced by Lactobacillus fermentum,Saccharomyces cerevisiae and Candida krusei in single starter culture fermentations of Ghanaian maize dough

AIMS: To identify and compare the volatile compounds associated with maize dough samples prepared by spontaneous fermentation and by the use of added starter cultures in Ghana. METHODS AND RESULTS: The starter cultures examined were Lactobacillus fermentum, Saccharomyces cerevisiae and Candida krusei. For identification of aroma volatiles, extracts by the Likens-Nickerson simultaneous distillation and extraction technique were analysed by gas chromatography-mass spectrometry (GC-MS) and using a trained panel of four judges by GC-Olfactometry (GC-sniffing). Compounds identified by GC-MS in maize dough samples after 72 h of fermentation included 20 alcohols, 22 carbonyls, 11 esters, seven acids, a furan and three phenolic compounds. Of the total 64 volatile compounds, 51 were detected by GC-sniffing as contributing to the aroma of the different fermented dough samples. Spontaneously fermented maize dough was characterized by higher levels of carbonyl compounds while fermentations with added L. fermentum recorded the highest concentration of acetic acid. S. cerevisiae produced higher amounts of fusel alcohols and increasing levels of esters with fermentation time and C. krusei showed similarity to L. fermentum with lower levels of most volatiles identified. CONCLUSION: The present study has given a detailed picture of the aroma compounds in fermented maize and demonstrated that the predominant micro-organisms in fermented maize dough can be used as starter cultures to modify the aroma of fermented maize dough. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has documented the advantage of using starter cultures in African traditional food processing and provided a scientific background for introducing better controlled fermentations.     

Responses of Listeria monocytogenes to acid stress and glucose availability revealed by a novel combination of fluorescence microscopy and microelectrode ion-selective techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H(+) at either side of the bacterial membrane in real time and therefore to evaluate the rate of H(+) flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH(o)) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH(i)) and net H(+) fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH(i) remained above 7.0 within a pH(o) range of 4 to 6 and decreased below pH(o) 4. Above pH(o) 4, H(+) extrusion increased correspondingly, with the maximum value at pH(o) 5.5, and below pH(o) 4, a net H(+) influx was observed. Without glucose in the medium, the pH(i) decreased, and a net H(+) influx was observed below pH(o) 5.5. A high correlation (R = 0.75 to 0.92) between the pH(i) and net H(+) flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH(i) homeostasis in L. monocytogenes subjected to acid stress.     

Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.     

Evolution of Cyanobacteria by Exchange of Genetic Material among Phyletically Related Strains

The cyanobacterial radiation consists of several lineages of phyletically (morphologically and genetically) related organisms. Several of these organisms show a striking resemblance to fossil counterparts. To investigate the molecular mechanisms responsible for stabilizing or homogenizing cyanobacterial characters, we compared the evolutionary rates and phylogenetic origins of the small-subunit rRNA-encoding DNA (16S rDNA), the conserved gene rbcL (encoding d-ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit), and the less conserved gene rbcX. This survey includes four categories of phyletically related organisms: 16 strains of Microcystis, 6 strains of Tychonema, 10 strains of Planktothrix, and 12 strains of Nostoc. Both rbcL and rbcX can be regarded as neutrally evolving genes, with 95 to 100% and 50 to 80% synonymous nucleotide substitutions, respectively. There is generally low sequence divergence within the Microcystis, Tychonema, and Planktothrix categories both for rbcLX and 16S rDNA. The Nostoc category, on the other hand, consists of three genetically clustered lineages for these loci. The 16S rDNA and rbcLX phylogenies are not congruent for strains within the clustered groups. Furthermore, analysis of the phyletic structure for rbcLX indicates recombinational events between the informative sites within this locus. Thus, our results are best explained by a model involving both intergenic and intragenic recombinations. This evolutionary model explains the DNA sequence clustering for the modern species as a result of sequence homogenization (concerted evolution) caused by exchange of genetic material for neutrally evolving genes. The morphological clustering, on the other hand, is explained by structural and functional stability of these characters. We also suggest that exchange of genetic material for neutrally evolving genes may explain the apparent stability of cyanobacterial morphological characters, perhaps over billions of years.The current species diversity of the cyanobacterial radiation comprises several lineages of phyletically (morphologically and genetically) related organisms (26). An intriguing question is whether this reflects stability of cyanobacterial characters or whether the phyletic similarities originate from relatively recent common ancestors. Analyses of precambrian microfossils (superficially, hardly distinguishable from recent cyanobacteria) support the view of retention of cyanobacterial properties (1, 11, 28). However, on the basis of molecular data, a 2-billion-year-old mutual ancestor for prokaryotes has been suggested (5), implying that the similarities between the earliest records of cyanobacteria and present-day species do not reflect homologies but rather indicate analogies. In this context, the phyletically clustered groups may reflect a relatively recent divergence of the modern species.In this work we have addressed, by molecular evolutionary studies, the mechanisms responsible for conserving or homogenizing phyletical characters within groups of cyanobacteria. We investigated the evolutionary rates and origins for two genomic regions, by analyzing strains both within and among groups of phyletically related organisms. This was done by comparative analysis of the small-subunit rRNA-encoding DNA (16S rDNA), which is conserved by the RNA function (37), and the rbcLX region with both conserved and less conserved elements. The rbcLX region contains an intergenic spacer (with no identified functional units), the gene rbcX with a possible chaperonin-like function (18), and the 3′ end of rbcL (encoding the highly conserved d-ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit [LSU]) (23). We analyzed a data set consisting of four phyletically clustered cyanobacterial strain categories, as inferred from microscopic observations and 16S rDNA analysis (26, 31). The data set includes the Microcystis category (16 strains), consisting of unicellular organisms, the Tychonema (6 strains) and Planktothrix (10 strains) categories, which contain multicellular, filamentous organisms, and the Nostoc category (12 strains), which includes both morphologically and genetically slightly divergent organisms (26, 34). The strains in this last category share among other features the ability of cellular differentiation to produce heterocysts with nitrogenase activity.Our sequence data suggest an evolutionary model involving several events of gene transfer between phyletically closely related organisms but not between less related organisms. We propose that this gene transfer has led to the observed sequence homogeneity for the groups of related organisms and that exchange of genetic material stabilizes the function and structure of proteins encoded by neutrally evolving genes. Our gene transfer model may explain the similarity between the fossil and the recent species.