Beta Gamma Delta Research Articles

A major substrate for MPF: cDNA cloning and expression of polypeptide chain elongation factor 1 gamma from goldfish (Carassius auratus).

One of the eukaryotic polypeptide chain elongation factors, EF-1 beta gamma delta complex, is involved in polypeptide chain elongation via the GDP/GTP exchange activity of EF-1 alpha. In the complex, EF-1 gamma has been reported to be a major substrate for maturation promoting factor (MPF). Here, we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, EF-1 gamma from the goldfish ovary. The cloned cDNA was 1490 bp in length and encoded 442 amino acids. The deduced amino acid sequence was highly homologous to EF-1 gamma from other species. Although, the phosphorylation site identified in Xenopus EF-1 gamma was not conserved in the goldfish homologue, phosphorylation analysis showed that the goldfish EF-1 gamma was phosphorylated by MPF. We concluded that EF-1 gamma is a substrate for MPF during oocyte maturation in goldfish.

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI‐1

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.

The epitope detected by cytotoxic T lymphocytes against thymus leukemia (TL) antigen is TAP independent.

Thymus leukemia (TL) antigens belong to the family of MHC class Ib antigens. We have shown in our previous studies that they serve as transplantation antigens, and can be recognized by both TCR alpha beta and TCR gamma delta cytotoxic T lymphocytes (CTL) with TL but not H-2 restriction. Although TL are known to be expressed TAP independently, it is unclear whether peptide loading on TL molecules is necessary for the formation of CTL epitopes. In the present study, we first showed that TL expression is beta(2)-microglobulin (beta(2)m)-dependent but TAP1 independent by flow cytometric analysis of thymocytes from beta(2)m- or TAP1-deficient mice crossed with TL transgenic mice expressing Tla(a)-3-TL on their thymocytes. Subsequently, we investigated the epitope recognized by CTL derived from C3H mice immunized with skin from a transgenic mouse expressing T3(b)-TL ubiquitously. Bulk CTL lines against TL from primary mixed lymphocyte cultures showed comparable cytotoxicity against T3(b)-TL transfectants of TAP2-deficient murine RMA-S grown at 37 degrees C to that against those grown at 25 degrees C. Furthermore, TCR alpha beta and TCR gamma delta CTL clones against TL recognized TL expressed on T3(b)-TL transfectants of RMA-S and Drosophila melanogaster cells having broad defects in peptide loading of MHC, and lysed these target cells. These results together indicate that TL-specific CTL populations primarily recognize epitopes expressed TAP independently.

On the prosthetic groups of the NiFe sulfhydrogenase from Pyrococcus furiosus: topology, structure, and temperature-dependent redox chemistry.

The sulfhydrogenase complex of Pyrococcus furiosus is an alpha beta gamma delta heterotetramer with both hydrogenase activity (borne by the alpha delta subunits) and sulfur reductase activity (carried by the beta gamma subunits). The beta-subunit contains at least two [4Fe-4S] cubanes and the gamma-subunit contains one [2Fe-2S] cluster and one FAD molecule. The delta-subunit contains three [4Fe-4S] cubanes and the alpha-subunit carries the NiFe dinuclear center. Only three Fe/S signals are observed in EPR-monitored reduction by dithionite, NADPH, or internal substrate upon heating. All other clusters presumably have reduction potentials well below that of the H+/H2 couple. Heat-induced reduction by internal substrate allows, for the first time, EPR monitoring of the NiFe center in a hyperthermophilic hydrogenase, which passes through a number of states, some of which are similar to states previously defined for mesophilic hydrogenases. The complexity of the observed transitions reflects a combination of temperature-dependent activation and temperature-dependent reduction potentials.

Downregulation of innate and acquired antitumor immunity by bystander gammadelta and alphabeta T lymphocytes with Th2 or Tr1 cytokine profiles.

It has been thought that natural killer (NK) cells appearing early in tumor lesions play a pivotal role in the innate immunity against tumor cells. Although NK cells serve as the first tumoricidal effector cells, they subsequently promote a shift in effectors from themselves to tumor-specific cytotoxic T lymphocytes (CTLs) that mediate the acquired immunity. The mechanism of this shift has not been fully elucidated, however, NK cell-derived T helper (Th) 1 cytokines such as interferon (IFN)-gamma seem to play a key role. Another NK-lineage, termed natural killer T (NK T) cells, may also participate in the innate period when they acquire the ability to secrete Th1 cytokines. Interleukin-4 (IL-4) and IL-10, belonging to Th2, and transforming growth factor-beta (TGF-beta), belonging to T regulatory (Tr) 1 cytokines, are known to suppress the development of NK, NK T cells, as well as CTLs and to block Th0 cell differentiation to Th1 cells, suggesting that tumor cells can evade the innate and acquired immunity by virtue of cells producing these inhibitory cytokines. In early tumor lesions of murine B16 melanoma, gammadelta T and alphabeta intermediate (int) T cells that co-infiltrate with NK and NK T cells can produce Th2 cytokines and inhibit the innate immunity. In MM2 mammary tumor-bearing mice, gammadelta T cells appearing both lesionally and systemically secrete Tr1-type cytokines and depress the acquired immunity. These Th2- or Tr1-type gammadelta T and alphabeta(int) T cells downregulate the tumoricidal cells by means of both their secreted cytokines and express major histocompatibility complex (MHC) class I molecules.

Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex.

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (alpha beta gamma delta)4. Previous studies have revealed that the activity of its catalytic gamma subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb gamma79) has been generated against isolated gamma subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the gamma subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the gamma-calmodulin binary complex, and the free gamma subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb gamma79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the gamma subunit containing the epitope for mAb gamma79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb gamma79, as well as the binding of mAb gamma79 to immobilized gamma subunit. Thus, Mg2+ increases the accessibility of the mAb gamma79 epitope in both the isolated gamma subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the gamma subunit.

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae.

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.

Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli.

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35%. Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively. The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem. 272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross-linking and blue native PAGE. P2X1 and P2X3 were N-terminally tagged with six histidine residues to allow for non-denaturing receptor isolation from cRNA-injected, [35S]methionine-labeled oocytes. The His-tag did not change the electrophysiological properties of the P2X1 receptor. His-P2X1 was found to carry four N-glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3, 3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) cross-linked digitonin-solubilized His-P2X1 and His-P2X3 quantitatively to homo-trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl-beta-D-maltoside migrated entirely as non-covalently linked homo-trimers, whereas the alpha2 beta gamma delta nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His-P2X1 was also identified as a homo-trimer. If n-octylglucoside was used for P2X receptor solubilization, homo-hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure. blue native PAGE/cross-linking/P2X receptor/quaternary structure.

Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding.

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.

Evidence that the same gamma delta T cells respond during infection-induced and autoimmune inflammation.

Inflammatory responses are induced in both testes of a mouse following injection of Listeria monocytogenes into one testis. Although the uninjected testis contains no detectable bacteria, it undergoes an autoimmune attack. Normally, the testis lacks lymphocytes, but in the infected and autoimmune state, both gamma delta and alpha beta T cells are found as infiltrates. Here, we have examined the repertoire of the infiltrating gamma delta T cells, using two different methods, and found a high frequency of V gamma 6/V delta 1 gamma delta T cells in both infected and autoimmune testes. All of these expressed the invariant V gamma 6/V delta 1 TCR previously reported. However, secondary gamma and delta transcripts present within V gamma 6/V delta 1 hybridomas indicated nonclonality. Interestingly, some of these secondary transcripts were derived from gamma gene rearrangements not previously found in this gamma delta T cell subset, implying a difference in its origin. The increase in V gamma 6/V delta 1 cells observed here in both infected and autoimmune testes, together with our previous finding of a preferential response by the same subset in Listeria-infected liver, indicates that their response is triggered by the inflammation rather than by the infectious agent or because they are already resident in the tissue. We and others have previously reported that the presence of gamma delta T cells during certain inflammatory conditions correlates with less host tissue damage. This result, together with the evidence presented here, further implies that a response by the V gamma 6/V delta 1 subset in some way exerts a controlling influence on the host inflammatory response.

Sequential development of intraepithelial gamma delta and alpha beta T lymphocytes expressing CD8 alpha beta in neonatal rat intestine: requirement for the thymus.

Previous studies in congenitally athymic nude rats have suggested that the thymus is important for the development of intestinal T cells. Here we have examined the effect of the nude mutation on intraepithelial lymphocyte (IEL) development from the perinatal period. By immunohistochemistry it was shown that CD3(-)CD8 alpha alpha + putative IEL precursors colonized the epithelium of both normal and athymic neonatal rats. Mature T cells, however, did not develop in athymic neonates. In normal rats, gamma delta T cells were present at birth and alpha beta T cells appeared within 8 days of postnatal life. At this age, the composition and relative number of intraepithelial T cells were similar to that in normal adult rats, with the exception that most neonatal T-cell receptor-gamma delta + and -alpha beta + IEL expressed CD8 beta. By contrast, extrathymic T-cell maturation in the gut of congenitally athymic rats occurred slowly, as CD3+ IEL did not appear until 4-6 months of age. These intraepithelial T cells displayed variable phenotypes and appeared to be induced by environmental antigens as they were not found in isolator-kept old nudes. In conclusion, the present results indicate that the major colonization of the gut epithelium with gamma delta and alpha beta T cells expressing CD8 alpha beta takes place perinatally and requires the presence of the thymus. The developmental relationship between these neonatal T cells and more immature CD3- CD8 alpha alpha +/- IEL remains elusive.

Molecular cloning of the breakpoints of the hereditary persistence of fetal hemoglobin type-6 (HPFH-6) deletion and sequence analysis of the novel juxtaposed region from the 3' end of the beta-globin gene cluster.

Naturally occurring deletion mutations within the human beta-globin cluster lead to specific, phenotypically discrete syndromes (i.e., delta beta-thalassemias and hereditary persistence of fetal hemoglobin, HPFH), characterized by increased production of fetal hemoglobin in adult life. We have previously characterized an enhancer element, which is juxtaposed to the fetal G gamma-gene, by means of a deletion first described in a Thai family. To obtain further insights into the mechanisms involved in this deletion, we have now characterized several of its novel features. Following amplification by the polymerase chain reaction and sequencing of the 1.5-kb bridging fragment, we have shown that the 5' breakpoint of the deletion occurs 1260 bp 3' of the fetal G gamma-globin gene, whereas the 3' breakpoint lies 521 bp upstream of the EcoRI site of the enhancer element and 2845 bp upstream of the 3' breakpoint of the Chinese (A gamma delta beta) zero-thalassemia deletion. The total length of the deletion is 101 kb, which resembles that of HPFH-1 and HPFH-2 deletions and a set of two gamma delta beta-thalassemia deletions. Our data further support the hypothesis that these sets of large deletions with almost identical lengths are generated via the loss of a complete chromatin loop. To elucidate further the mechanisms leading to the deletion, we have sequenced the novel 0.5-kb region residing immediately 3' to the breakpoint and shown that it contains putative binding sites for several transcription factors, such as HNF-1, AP-1, and TFIID. Sequence comparison of the deletion breakpoints reveals no junctional homology, indicating an end-to-end joining of blunted ends; a pair of 7-nt complementary repeats adjacent to a set of a direct CCCT repeat flanks the breakpoints. This limited homology constitutes a frequent characteristic of a non-homologous recombination mechanism. All these features of the HPFH-6 deletion suggest that this mutation has resulted from a non-homologous recombination event.

N-Glycosylation at the conserved sites ensures the expression of properly folded functional ACh receptors

The role of the conserved carbohydrate moiety in the expression of complete acetylcholine receptor (AChR), alpha2 beta gamma delta was re-investigated by expressing additional site-directed mutant subunits, lacking an N-glycosylation site, in Xenopus oocytes. All mutant subunits were stably expressed and appeared to associate with other normal subunits; however, removal of carbohydrate on the alpha subunit inhibited the formation of 125I-alpha-bungarotoxin (alpha-BuTX) binding sites and functional ACh-gated ion channels. 125I-alpha-BuTX binding to AChRs was also significantly reduced by removal of the conserved carbohydrate on the gamma or delta subunits. Immunoprecipitation with monoclonal antibodies that recognize the two distinct alpha-BuTX sites on the AChR indicated that the mutant gamma subunit did not interfere with efficient formation of the alpha-BuTX binding site at the alpha/delta interface, but loss of the carbohydrate did interfere with formation of the alpha-BuTX binding site at the alpha/mutant gamma interface. A similar result was obtained with the mutant delta subunit. Furthermore, the mutant gamma and mutant delta subunits were not incorporated efficiently into the mature (correct tertiary conformation capable of alpha-BuTX binding) alpha beta delta or alpha beta gamma complexes, respectively. Since both mutant gamma and mutant delta subunits were capable of assembling with the alpha subunits (immature assembly), these results suggest that the formation of the two alpha-BuTX binding sites requires correct folding of the alpha gamma and alpha delta complexes, which is aided by the conserved carbohydrate on the gamma and delta subunits. Electrophysiological experiments demonstrated that functional receptors containing mutant subunits were produced, but the functional properties of the mutant receptors were differentially altered, depending on the subunit mutated. Together, our results suggest that N-glycosylation of AChR subunits ensures the correct folding of important functional domains and expression of proper functional receptors in the plasma membrane.

TCR gene recombination and alpha beta-gamma delta lineage divergence: productive TCR-beta rearrangement is neither exclusive nor preclusive of gamma delta cell development.

Two types of T lymphocytes can be generated intrathymically, distinguishable by either TCR-gamma delta or -alpha beta surface expression. Regulation of the intrathymic divergence of these cells is unresolved, at least in part because thymically derived gamma delta cells have rarely been studied. We used quantitative Southern blotting together with PCR-based cloning/sequencing and restriction fragment length polymorphism to analyze TCR-alpha and -beta gene recombination in thymically derived gamma delta cells. We found that TCR-beta gene recombination is a frequent occurrence in thymic gamma delta cells. Furthermore, not only do complete (V-D-J) TCR-beta gene rearrangements occur in thymic gamma delta cells, but the frequency of in-frame rearrangements is greater than would be predicted based upon random occurrence. In contrast, we show that thymically derived gamma delta cells do not make detectable rearrangements of the TCR-alpha locus. These studies clearly demarcate a point for alpha beta vs gamma delta commitment in the thymus, after TCR-beta but before TCR-alpha gene recombination. Further, while our data support gamma delta lineage commitment as a consequence of successful TCR-gamma and -delta gene rearrangement, we do not find support for a competitive model of lineage commitment, since productive TCR-beta gene rearrangement does not necessarily relegate cells to the alpha beta lineage.

Melanoma specific Th1 cytotoxic T lymphocyte lines in Vogt-Koyanagi-Harada disease.

To determine the functional properties and cytokine production profiles of melanoma specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood leucocytes of two patients with Vogt-Koyanagi-Harada disease (VKH). Melanoma specific CTL lines were established by long term coculture with a human melanoma cell line (P-36). Cytotoxic activity against P-36 was measured by 51Cr release. The involvement of human leucocyte antigen (HLA) class I or class II molecules in the cytotoxicity of the CTL lines against P-36 was analysed using anti-HLA class I or anti-HLA class II monoclonal antibody (MAb). Surface molecules of CTL lines were analysed by flow cytometry using MAbs specific for CD4, CD8, CD16, CD25, CD56, HLA-DR, T cell antigen receptor (TCR) alpha beta and TCR gamma delta. Cytokine production and soluble interleukin 2 receptor (sIL-2R) secretion were determined by enzyme linked immunosorbent assays. mRNAs of cytokines were analysed using reverse transcription polymerase chain reaction (RT-PCR). CTLs showed strong cytotoxic activity against P-36. The CTL activity of the cell lines against P-36 was inhibited by the anti-HLA-DR MAb, whereas the MAb specific for monomorphic determinants of HLA-A, B, and C failed to block lytic activity. Flow cytometry identified the following surface molecules: CD4+, CD8-, CD16-, CD25+, CD56-, HLA-DR+, TCR alpha beta +, and TCR gamma delta-. CTLs constitutively produced a high level of IL-6. IL-6 production and sIL-2R secretion of CTLs were enhanced when CTLs were stimulated with P-36. CTLs also produced high levels of interferon gamma (IFN-gamma) and IL-2, but not IL-4. mRNAs of IL-2 and IFN-gamma were detected by RT-PCR in the CTLs. Melanoma specific HLADR restricted T helper 1 (Th1) CTLs may play a role in the immunopathogenesis of VKH.

Thymocyte emigration in the chicken: an over-representation of CD4+ cells over CD8+ in the periphery.

We have examined the emigration of chicken thymocytes after intrathymic fluorescein isothiocyanate (FITC) labelling in situ. In this paper we show that in young birds about 0.7% and 0.4% of thymocytes emigrate from the thymus to the blood and the spleen, respectively, per day. This suggests that, as in mammals, most thymocytes die within the thymus. At 3 weeks of age gamma delta and alpha beta T cells leave the thymus in comparable levels to their appearance in the blood. The phenotype of recent emigrants in peripheral tissues is similar to that of mature T cells. Interestingly, recent emigrants contain relatively much higher numbers of CD4+ and fewer CD8+ cells than is observed in peripheral tissues in a steady-state situation.

Binding sites contribute unequally to the gating of mouse nicotinic alpha D200N acetylcholine receptors.

1. Single channel currents were recorded from HEK 293 cells expressing recombinant mouse adult (alpha 2 beta delta gamma) acetylcholine receptors (AChRs) containing a mutation at residue D200 of the alpha-subunit. Rate and equilibrium constants for AChR activation were estimated from open and closed time obtained over a range of ACh concentrations. 2. Mutation of alpha D200 to asparagine (alpha D200N) dramatically slows the rate constant of channel opening, with adult AChRs slowing 100-fold and embryonic AChRs slowing 400-fold. the rate constant of channel closing increased 3-fold, resulting in a decrease of the gating equilibrium constant of up to 1200-fold. In contrast to channel gating steps, ACh-binding steps are only modestly effected by alpha D200N. 3. Introduction of a potential glycosylation site in alpha D200N cannot account for the effect on channel gating because eliminating the consensus for glycosylation with the mutation alpha D200N + T202V fails to restore efficient gating. Gating is similarly impaired with the substitutions of E, K and Q at position alpha 200. 4. the agonists carbamylcholine and tetramethylammonium also activate the alpha D200N AChR, but with channel opening rates even slower than with ACh. The agonist dependence of the opening rate constant is similar in alpha D200N and wild type AChRs. 5. AChRs containing D200N at just one of the two alpha-subunits show either small or large changes in the gating equilibrium constant, presumably due to the presence of the mutation at either the alpha delta or alpha epsilon/alpha gamma sites. The changes in free energy of channel gating show that the contribution of each binding site is nearly independent. However, the sites do not contribute equally to gating, as an alpha D200N mutation at the alpha epsilon or alpha gamma binding site slows channel opening relatively more than at the alpha delta site.

Increased division of alpha beta TCR+ and gamma delta TCR+ intestinal intraepithelial lymphocytes after oral administration of cholera toxin.

Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5-bromo-2'-deoxyuridine (BRdU) and membrane alpha beta T-cell receptors (TCR) or gamma delta TCR staining in frozen sections. Cholera toxin (10 micrograms) produced a two- to eightfold-increase in the uptake of BRdU in alpha beta TCR+ IEL in the duodenum and a two-to fivefold increase in gamma delta TCR IEL in the ileum. Increased uptake of BRdU was also seen after a dose of 100 micrograms of CT but this dose was also associated with the loss of alpha beta TCR+ IEL and gamma delta TCR+ IEL in the duodenum. CT-A and CT-B subunit produced increased BRdU incorporation by alpha beta TCR in the duodenum and by gamma delta TCR IEL in the ileum. Cholera toxin therefore appears to be mitogenic for IEL probably due to an indirect mechanism.

Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells.

Bipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR) alpha beta and TCR gamma delta cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells. The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown. In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process. The IL-15 receptor is composed of the gamma and beta chains of the IL-2 receptor (IL-2R gamma and IL-2R beta) and of a specific alpha chain (IL-15R alpha). Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes. IL-2R beta-expressing cells were present in the fetal thymus with a CD25-CD44+Fc gamma R+HSA-/low TCR- phenotype, which is characteristic of progenitor cells. These cells also expressed IL-15R alpha messenger RNA. Sorted IL-2R beta + TCR- cells differentiated into TCR alpha beta and TCR gamma delta cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells. This shows that IL-2R beta +TCR- cells of the fetal thymus contain bipotential T/NK progenitors. Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations. The largest expansion occurred in the TCR gamma delta compartment. In contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCR gamma delta cells. High concentrations of IL-15 blocked TCR alpha beta development and shifted differentiation towards NK cells. Differentiation towards TCR gamma delta cells still proceeded. High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures. Importantly, blocking of IL-2R alpha in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2R alpha negatively regulates cell expansion. Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.