Structure of the T cell antigen receptor (TCR): two CD3 epsilon subunits in a functional TCR/CD3 complex.

Transgenic mice carrying and expressing the human CD3 epsilon gene incorporate the corresponding protein product into T cell receptor (TCR)/CD3 complexes on thymocyte and T cell surfaces. The chimeric antigen receptors allow normal T cell development and selection of repertoires in vivo and are able to transduce activation signals in vitro. We have exploited the ability to distinguish mouse (m) and human (h)CD3 epsilon chains to analyze the stoichiometry of CD3 epsilon in transgenic mouse TCRs. Immunoprecipitation and fluorescence resonance energy transfer experiments demonstrate that such TCRs can contain both h- and mCD3 epsilon chains, implying that more than one CD3 epsilon subunit occurs per TCR. Antigen comodulation studies are consistent with a stochastic use of h- or mCD3 epsilon during receptor assembly, and further suggest a structure for the TCR/CD3 complex with two CD3 epsilon chains. The determination of CD3 epsilon subunit stoichiometry, together with existing biochemical data, allows the generation of a minimal model for the structure of the TCR and illustrates the potential value of the transgenic approach to the analysis of complex receptors.

In vivo persistence of expanded clones specific for bacterial antigens within the human T cell receptor alpha/beta CD4-8- subset.

We analyzed the T cell receptor (TCR) rearrangements of 100 TCR-alpha/beta CD4-CD8- (double negative [DN]) T cell clones from normal individuals. We found that in four out of six donors this subset contains expanded clones that often account for 0.5% and, in one individual, even 7% of all peripheral blood lymphocytes. By combining limiting dilution analysis and N region oligotyping of polymerase chain reaction amplified TCR cDNA, we could measure the clonal size and show that two of these expanded clones remain stable in size for up to 4 yr in peripheral blood. The expanded clones analyzed ex vivo are not cycling and CD45 RAhi ROlo, but express high levels of alpha 4/beta 1 integrins, suggesting that they may have reverted to resting cells after activation. One of these expanded DN clones proliferates in vitro in response to Escherichia coli presented by monocytes cultured in GM-CSF plus IL-4 and kills CD1a+ Molt-4 cells. In contrast to what was found in the alpha/beta DN subset, alpha/beta CD4+ T cell clones specific for a tetanus toxin epitope showed a very small clonal size (< 1 in 10(7)) and could not be reisolated after 2 yr. Taken together, these results indicate that large clonal size and persistence are distinctive features of alpha/beta DN cells specific for bacterial antigens. These cells may use antigen-presenting cells, restriction molecules, and selection routes different from those used by antigen-specific CD4+ T cells.

Rapid method for isolation of desiccation-tolerant strains and xeroprotectants.

A novel biotechnological process has been developed for the isolation of desiccation-tolerant microorganisms and their xeroprotectants, i.e., compatible solutes involved in long-term stability of biomolecules in the dry state. Following exposure of soil samples to chloroform, we isolated a collection of desiccation-tolerant microorganisms. This collection was screened for the production of xeroprotectants by a variation of the bacterial milking (osmotic downshock) procedure and by a novel air-drying/rehydration ("dry milking") incubation method. The resultant solutes were shown to protect both proteins and living cells against desiccation damage, thereby validating them as xeroprotectants. Nuclear magnetic resonance (NMR) analytical studies were performed to identify the xeroprotectants; synthetic mixtures of these compounds were shown to perform similarly to natural isolates in drying experiments with proteins and cells. This new approach has biotechnological and environmental implications for the identification of new xeroprotectants of commercial and therapeutic value.

Expression of simian virus 40 large T antigen in embryonal carcinoma cell hybrids.

Previous work has shown that murine embryonal carcinoma cells are refractory to infection with various viruses, including simian virus 40. Thus, large T and small t antigens, the products of the simian virus 40 early region, are not produced when the virus infects embryonal carcinoma cells, in contrast to other cell types. We show, by qualitative and quantitative analyses, that embryonal carcinoma cell hybrids, containing a simian virus 40 early region integrated into human DNA, are capable of producing viral large T antigen.

Dehydration-induced tps gene transcripts from an anhydrobiotic nematode contain novel spliced leaders and encode atypical GT-20 family proteins.

Accumulation of the non-reducing disaccharide trehalose is associated with desiccation tolerance during anhydrobiosis in a number of invertebrates, but there is little information on trehalose biosynthetic genes in these organisms. We have identified two trehalose-6-phosphate synthase (tps) genes in the anhydrobiotic nematode Aphelenchus avenae and determined full length cDNA sequences for both; for comparison, full length tps cDNAs from the model nematode, Caenorhabditis elegans, have also been obtained. The A. avenae genes encode very similar proteins containing the catalytic domain characteristic of the GT-20 family of glycosyltransferases and are most similar to tps-2 of C. elegans; no evidence was found for a gene in A. avenae corresponding to Ce-tps-1. Analysis of A. avenae tps cDNAs revealed several features of interest, including alternative trans-splicing of spliced leader sequences in Aav-tps-1, and four different, novel SL1-related trans-spliced leaders, which were different to the canonical SL1 sequence found in all other nematodes studied. The latter observation suggests that A. avenae does not comply with the strict evolutionary conservation of SL1 sequences observed in other species. Unusual features were also noted in predicted nematode TPS proteins, which distinguish them from homologues in other higher eukaryotes (plants and insects) and in micro-organisms. Phylogenetic analysis confirmed their membership of the GT-20 glycosyltransferase family, but indicated an accelerated rate of molecular evolution. Furthermore, nematode TPS proteins possess N- and C-terminal domains, which are unrelated to those of other eukaryotes: nematode C-terminal domains, for example, do not contain trehalose-6-phosphate phosphatase-like sequences, as seen in plant and insect homologues. During onset of anhydrobiosis, both tps genes in A. avenae are upregulated, but exposure to cold or increased osmolarity also results in gene induction, although to a lesser extent. Trehalose seems likely therefore to play a role in a number of stress responses in nematodes.

Multiple horizontally acquired genes from fungal and prokaryotic donors encode cellulolytic enzymes in the bdelloid rotifer Adineta ricciae.

The bdelloid rotifer, Adineta ricciae, an anhydrobiotic microinvertebrate, exhibits a high rate of horizontal gene transfer (HGT), with as much as 10% of its transcriptome being of foreign origin. Approximately 80% of these foreign transcripts are involved in metabolic processes, and therefore bdelloids represent a useful model for assessing the contribution of HGT to biochemical diversity. To validate this concept, we focused on cellulose digestion, an unusual activity in animals, which is represented by at least 16 genes encoding cellulolytic enzymes in A. ricciae. These genes have been acquired from a variety of different donor organisms among the bacteria and fungi, demonstrating that bdelloids use diverse genetic resources to construct a novel biochemical pathway. A variable complement of the cellulolytic gene set was found in five other bdelloid species, indicating a dynamic process of gene acquisition, duplication and loss during bdelloid evolution. For example, in A. ricciae, gene duplications have led to the formation of three copies of a gene encoding a GH45 family glycoside hydrolase, at least one of which encodes a functional enzyme; all three of these gene copies are present in a close relative, Adineta vaga, but only one copy was found in each of four Rotaria species. Furthermore, analysis of expression levels of the cellulolytic genes suggests that a bacterial-origin cellobiase is upregulated upon desiccation. In summary, bdelloid rotifers have apparently developed cellulolytic functions by the acquisition and domestication of multiple foreign genes.

Cloning and chromosomal localization of the human BARX2 homeobox protein gene.

The human BARX2 gene encodes a homeodomain-containing protein of 254 amino acids, which binds optimally to the DNA consensus sequence YYTAATGRTTTTY. BARX2 is highly expressed in adult salivary gland and is expressed at lower levels in other tissues, including mammary gland, kidney, and placenta. The BARX2 gene consists of four exons, and is located on human chromosome 11q25. This chromosomal location is within the minimal deletion region for Jacobsen syndrome, a syndrome including craniosynostosis and other developmental abnormalities. This chromosomal location, along with the reported expression of murine barx2 in craniofacial development, suggests that BARX2 may be causally involved in the craniofacial abnormalities in Jacobsen syndrome.

Intracellular trehalose improves osmotolerance but not desiccation tolerance in mammalian cells.

Trehalose has been shown to play a role in osmotolerance or desiccation tolerance in some microorganisms, anhydrobiotic invertebrates and resurrection plants. To test whether trehalose could improve stress responses of higher eukaryotes, a mouse cell line was genetically engineered to express bacterial trehalose synthase genes. We report that the resulting levels of intracellular trehalose ( approximately 80 mM) are able to confer increased resistance to the partial dehydration resulting from hypertonic stress, but do not enable survival of complete desiccation due to air drying.

Bacterial toxin RelE induces apoptosis in human cells.

The bacterial protein RelE severely restricts prokaryotic cell growth, probably by acting as a global inhibitor of translation. It is ubiquitous in prokaryotes as part of the RelE-RelB toxin-antitoxin system, and may be activated by nutritional stress. When the relE gene from Escherichia coli was expressed inducibly in a human osteosarcoma cell line, it was shown to retard growth and to lead to cell death by apoptosis. RelE is therefore unusual among bacterial toxins in possessing broad activity against both prokaryotes and eukaryotes, perhaps by acting on evolutionarily conserved components of the translation machinery.

Production and secretion of recombinant soluble CD3 polypeptides by myeloma-derived transfectant clones.

Soluble forms of three human CD3 proteins have been produced by recombinant DNA techniques. The extracellular domain of CD3-gamma, -delta or -epsilon has been linked to the constant region of mouse immunoglobulin kappa light chain to form gamma-kappa, delta-kappa and epsilon-kappa chimaeric proteins. These are secreted by mouse myeloma-derived transfectant cell lines and are immunoprecipitable by CD3- or kappa-specific polyclonal antisera. Yields of 100-500 micrograms secreted recombinant proteins per litre of culture medium were obtained, which could be purified by anti-kappa affinity chromatography. The production of soluble CD3 illustrates the applicability of this technology to a loosely associated protein complex.

Most Jacobsen syndrome deletion breakpoints occur distal to FRA11B.

Recent studies have identified a (CCG)n repeat in the 5' untranslated region of the CBL2 protooncogene (11q23.3) and have demonstrated that expansion of this repeat causes expression of the folate-sensitive fragile site FRA11B. It has also been demonstrated that FRA11B is the site of breakage in some cases of Jacobsen syndrome (JS) involving terminal deletions of chromosome 11q. We report on 2 patients with JS and a 46,XX,del(11)(q23.3) karyotype. In both cases, microsatellite and fluorescence in situ hybridization analyses indicated that the deletion breakpoint was approximately 1.5-3 Mb telomeric to FRA11B. There was no evidence of expansion of the CBL2 (CCG)n repeat in the parents of either patient. The deleted chromosome was of paternal origin in both cases, although it was of maternal origin in the cases reported to be caused by FRA11B. These findings and those in previously reported patients suggest that the breakpoint for most 11q deletions in JS patients is telomeric to FRA11B, which raises the possibility that there may be other fragile sites in 11q23.3 in addition to FRA11B. These findings also support previous evidence that there may be a propensity for breakpoints to differ depending on the parental origin of the deleted chromosome.

Reduction of suspended biomass in municipal wastewater using bdelloid rotifers.

Clarification of municipal wastewater was shown to be improved significantly by the addition of cultured bdelloid rotifers. The rate and degree of suspended particle removal were correlated with rotifer number. The size range of unsettled particles suspended in wastewater was determined and found to overlap with the size range of particles consumed by rotifers. Rotifers were shown to have two distinct effects on suspended particles: consumption of biomass due to feeding activity; and improved settling, probably due to enhanced aggregation. These experiments demonstrate the potential for the use of bdelloid rotifers in an enhanced wastewater treatment process, with reduced biomass production and improved effluent clarity.

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.

Physical mapping of the CD3 gene cluster.

By a combination of pulsed-field gel electrophoresis and molecular cloning techniques, the human CD3-gamma, CD3-delta and CD3-epsilon genes have been shown to be clustered in approximately 50 kb of chromosome 11q23. The mouse genes are probably organised in a similar fashion on chromosome 9. An approach to the construction of a long-range physical map surrounding the CD3 gene cluster is described, which will complement the genetic maps of the region.

Distinct breakpoints in band 11q23 of the t(4;11) and t(11;14) associated with leukocyte malignancy.

Several non-random translocation breakpoints associated with leukemia or lymphoma have been shown to occur in chromosome band 11q23 between the genes CD3G and PBGD, a distance of approximately 750 kb. A combination of yeast artificial chromosome (YAC) cloning, in situ hybridization, and pulsed field gel electrophoresis (PFGE) experiments has further refined the interval containing one of these breakpoints, t(4;11)(q21;q23), to within 200 kb of CD3G. We have extended the PFGE analysis to show that the t(4;11) breakpoint lies in a region of approximately 100 kb, situated 100 kb distal to CD3G. Furthermore, we show that a second 11q23 breakpoint, t(11;14)(q23;q32), which was also previously mapped between CD3G and PBGD, is distinct from that of the t(4;11) chromosome. The 11q23 sequences that are involved at the t(11;14) breakpoint are not present in a YAC containing the t(4;11) breakpoint. The t(11;14) breakpoint has been localized on the PFGE map of the CD3G-PBGD interval and is at least 110 kb distal to the t(4;11) breakpoint, thus demonstrating heterogeneity among 11q23 breakpoints.

Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis.

In 1702, Van Leeuwenhoek was the first to describe the phenomenon of anhydrobiosis in a species of bdelloid rotifer, Philodina roseola. It is the purpose of this review to examine what has been learned since then about the extreme desiccation tolerance in rotifers and how this compares with our understanding of anhydrobiosis in other organisms. Remarkably, much of what is known today about the requirements for successful anhydrobiosis, and the degree of biostability conferred by the dry state, was already determined in principle by the time of Spallanzani in the late 18th century. Most modern research on anhydrobiosis has emphasized the importance of the non-reducing disaccharides trehalose and sucrose, one or other sugar being present at high concentrations during desiccation of anhydrobiotic nematodes, brine shrimp cysts, bakers' yeast, resurrection plants and plant seeds. These sugars are proposed to act as water replacement molecules, and as thermodynamic and kinetic stabilizers of biomolecules and membranes. In apparent contradiction of the prevailing models, recent experiments from our laboratory show that bdelloid rotifers undergo anhydrobiosis without producing trehalose or any analogous molecule. This has prompted us to critically re-examine the association of disaccharides with anhydrobiosis in the literature. Surprisingly, current hypotheses are based almost entirely on in vitro data: there is very limited information which is more than simply correlative in the literature on living systems. In many species, disaccharide accumulation occurs at approximately the same time as desiccation tolerance is acquired. However, several studies indicate that these sugars are not sufficient for anhydrobiosis; furthermore, there is no conclusive evidence, through mutagenesis or functional knockout experiments, for example, that sugars are necessary for anhydrobiosis. Indeed, some plant seeds and micro-organisms, like the rotifer, exhibit excellent desiccation tolerance in the absence of high intracellular sugar concentrations. Accordingly, it seems appropriate to call for a re-evaluation of our understanding of anhydrobiosis and to embark on new experimental programmes to determine the key molecular mechanisms involved.

Plastic encapsulation of stabilized Escherichia coli and Pseudomonas putida.

Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials. Bacteria are recovered by rehydration after physical disruption of the plastic. P. putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination.

The effect of horizontal differential prism on the binocular contrast sensitivity function.

The photopic binocular contrast sensitivity function is measured using a two-way resolution choice sinusoidal-grating target with horizontal differential prism before the right eye of one subject (AHT). A significant drop in sensitivity occurred with 3 prism-dioptres base-out or 4 prism-dioptres base-in prism at an observation distance of 5.9 m, and with 2 prism-dioptres base-out or 8 prism-dioptres base-in at 50 cm. An equivalent effect was confirmed with three additional subjects at a test distance of 2 m. The relationship between these results, the passive position, and the fusional reserves is discussed and reference is made to the effect of vertical differential prism.

Thymocyte clones from 14-day mouse embryos. II. Transcription of T3 gamma gene may precede rearrangement of TcR delta and expression of T3 delta, T3 epsilon and T11 genes.

We have studied the state of TcR delta gene and the expression of T3 delta, T3 epsilon, T3 gamma and T11 (CD2) genes in the fetal thymocyte (FT) clones A2, G15, H5, E10, D5, H12, F1 and D11 obtained from a 14-day B10.BR mouse fetal thymus. The eight FT clones contain the TcR delta gene in the germ-line configuration as determined by Southern blot analysis. With the exception of clones E10 and D5, the other six FT clones express normal sized transcripts for T3 gamma gene and none of the eight FT clones produced detectable RNA transcripts for T3 delta, T3 epsilon and T11 genes as assessed by Northern blot analysis. Together with our previous work showing that all eight FT clones contain the TcR gamma and the TcR beta gene clusters in the germ-line configuration, the data indicate that the FT clones represent the earliest stage of T cell development identified within the thymus. Our results provide evidence that (a) the T3 gamma gene is the first of the genes that encode components of a TcR/T3 complex to be expressed in ontogeny within the thymus; (b) the T3 (delta, epsilon, gamma) genes are switched on asynchronously and their expression must be differentially regulated, and (c) the T11 gene product may not be involved in early stages of T cell development within the thymus.