Search PubMed⌕ Search

Biomedical subjects

R Spoerl

Publications and source records attributed to R Spoerl.

18 recordsLinked to original sources
PubMed1999

Expression, purification, and functional analysis of murine ectodomain fragments of CD8alphaalpha and CD8alphabeta dimers.

Soluble mouse CD8alphaalpha and CD8alphabeta dimers corresponding to the paired ectodomains (CD8(f)) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.1 cells as secreted proteins using a leucine zipper strategy. The affinity of CD8alphaalpha(f) for H-2K(b) as measured by BIAcore revealed a approximately 65 microM K(d), similar to that of CD8alphabeta(f). Consistent with this result, CD8alphaalpha(f) as well as CD8alphabeta(f) blocked the effector function of N15 T cell receptor transgenic cytolytic T cells in a comparable, dose-dependent fashion. Furthermore, both Lec3.2.8.1-produced and Chinese hamster ovary-produced CD8 homodimers and heterodimers were active in the inhibition assay. These results suggest that the Ig-like domains of CD8 molecules are themselves sufficient to block the requisite transmembrane CD8-pMHC interaction between cytolytic T lymphocytes and target cells. Moreover, given the similarities in co-receptor affinities for pMHC, the findings suggest that the greater efficiency of CD8alphabeta versus CD8alphaalpha co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8alphaalpha, the yield, purity and homogeneity of the deglycosylated protein resulting from this expression system is sufficient for crystallization and x-ray diffraction at atomic resolution.

Animals↗
PubMed1999

The CD8beta ectodomain contributes to the augmented coreceptor function of CD8alphabeta heterodimers relative to CD8alphaalpha homodimers.

Within the lymphoid compartment, CD8 is expressed either as an alphaalpha homodimer or as an alphabeta heterodimer. Prior functional characterization of CD8alpha transfectants has demonstrated that CD8alphaalpha homodimers can reconstitute T cell responses in the absence of the CD8beta subunit. In order to now examine the role of CD8beta in TCR recognition, the CD8alpha cDNA alone or in combination with CD8beta cDNA was transfected into the mouse T cell hybridoma, N15wt, specific for VSV8/Kb. Comparison of antigen-induced IL-2 production reveals that CD8alphabeta+ transfectants are 100-fold more sensitive in molar terms of peptide than CD8alphaalpha+ transfectants. This enhancement of IL-2 production is independent of CD8alpha or CD8beta cytoplasmic tails as demonstrated by analysis of cytoplasmic deletion mutants CD8alpha'beta, CD8alphabeta', and CD8alpha'beta'. These results indicate that the ectodomain of the CD8beta chain greatly enhances the coreceptor function of the CD8alphabeta molecule, at least for certain class I MHC restricted alphabeta TCRs.

Animals↗
PubMed1998

Structural basis of CD8 coreceptor function revealed by crystallographic analysis of a murine CD8alphaalpha ectodomain fragment in complex with H-2Kb.

The crystal structure of the two immunoglobulin variable-like domains of the murine CD8alphaalpha homodimer complexed to the class I MHC H-2Kb molecule at 2.8 A resolution shows that CD8alphaalpha binds to the protruding MHC alpha3 domain loop in an antibody-like manner. Comparison of mouse CD8alphaalpha/H-2Kb and human CD8alphaalpha/HLA-A2 complexes reveals shared as well as species-specific recognition features. In both species, coreceptor function apparently involves the participation of CD8 dimer in a bidentate attachment to an MHC class I molecule in conjunction with a T cell receptor without discernable conformational alteration of the peptide or MHC antigen-presenting platform.

Amino Acid Sequence↗
PubMed1997

Topology of T cell receptor-peptide/class I MHC interaction defined by charge reversal complementation and functional analysis.

The molecular interactions between the CD8 co-receptor dependent N15 and N26 T cell receptors (TCRs) and their common ligand, the vesicular stomatitis virus octapeptide (VSV8) bound to H-2Kb, were studied to define the docking orientation(s) of MHC class I restricted TCRs during immune recognition. Guided by the molecular surfaces of the crystallographically defined peptide/MHC and modeled TCRs, a series of mutations in exposed residues likely contacting the TCR ligand were analyzed for their ability to alter peptide-triggered IL-2 production in T cell transfectants. Critical residues which diminished antigen recognition by 1000 to 10,000-fold in molar terms were identified in both N15 Valpha (alphaE94A or alphaE94R, Y98A and K99) and Vbeta (betaR96A, betaW97A and betaD99A) CDR3 loops. Mutational analysis indicated that the Rp1 residue of VSV8 is critical for antigen recognition of N15 TCR, but R62 of H-2Kb is less critical. More importantly, the alphaE94R mutant could be fully complemented by a reciprocal charge reversal at Kb R62 (R62E). This result suggests a direct interaction between N15 TCR Valpha E94R and Kb R62E residues. As Rp1 of VSV8 is adjacent to R62 in the VSV8/Kb complex and essential for T cell activation, this orientation implies that the N15 Valpha CDR3 loop interacts with the N-terminal residues of VSV8 with the Valpha domain docking to the Kb alpha2 helix while the N15 Vbeta CDR3 loop interacts with the more C-terminal peptide residues and the Vbeta domain overlies the Kb alpha1 helix. An equivalent orientation is suggested for N26, a second VSV8/Kb specific TCR. Given that genetic analysis of two different class II MHC-restricted TCRs and two crystallographic studies of class I restricted TCRs offers a similar overall orientation of V domains relative to alpha-helices, these data raise the possibility of a common docking mode between TCRs and their ligands regardless of MHC restriction.

Amino Acid Sequence↗
PubMed1997

Major histocompatibility complex recognition by immune receptors: differences among T cell receptor versus antibody interactions with the VSV8/H-2Kb complex.

The surface residues of the VSV8/Kb complex important for recognition by N15 and N26 alphabeta T cell receptors (TCR) were mapped by mutational analysis and compared to each other and with epitopes of well-characterized Kb specific monoclonal antibodies (mAb). Three features of immune receptor recognition emerge. First, the footprints of the two TCR on VSV8/Kb are similar with more than 80 % overlap between sites. Given that only 8 of 14 surface exposed VSV8/Kb residues identified as critical for TCR interaction are in common, the chemical basis of the N15 and N26 interactions is nevertheless distinct. Second, the cognate peptide is a major focus of TCR recognition: mutation at any of the three exposed side chains (at p1, p4 or p6) abrogates interaction of both TCR as measured by functional T cell activation. Third, in contrast to TCR, mAb bind to discrete segments on the periphery of the alpha1 and/or alpha2 helices without orientational restriction. These findings suggest that unlike soluble antibodies, surface membrane receptor-ligand interactions on opposing cells (i.e. TCR-peptide/ MHC, CD8-MHC) limit the orientational freedom of the TCR in the immune recognition process.

Animals↗
PubMed1996

Crystallization of a deglycosylated T cell receptor (TCR) complexed with an anti-TCR Fab fragment.

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the "Velcro" leucine zipper sequence to facilitate alpha-beta pairing. Upon secretion in culture media, the VSV-8-specific/H2-Kb-restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2-Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15(s), a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cbeta domain, was thrombin-cleaved and endoglycosidase H-digested, and the two derivatives were termed iN15DeltaH and N15(s)DeltaH, respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 alpha and beta subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cbeta-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15DeltaH and N15(s)DeltaH. Both N15(s)DeltaH-Fab[H57] and iN15DeltaH-Fab[H57] complexes crystallize, with the former diffracting to 2.8-A resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.

Animals↗
PubMed1981

[Nutritive effects of an intestinal alpha-glucosidases-inhibition in rats (author's transl)].

In a balance trial the effect of different dietary doses of the alpha-glucosidases-inhibitor Bay g5421 on nutrient digestibility and N-balance was tested with rats. The supplements of 30, 90 and 210 mg Bay g5421 per kg of feed reduced the digestibility of starch plus sucrose by 2, 16 and 27%. The protein digestibility decreased by 5, 14 and 19%. This shows that for the organism 3, 13 and 24% less digestible energy was available. The excretion of the urinary energy was not influenced in the different groups. The elevated fecal N excretion with the supplements of 30 and 90 mg Bay g5421 per kg of feed was compensated by a lower N-excretion in the urine. At the dose level of 210 mg Bay g5421 N balance was diminished.

Acarbose↗
PubMed1981

[Effect of inhibition of intestinal glucosidase on protein and energy balance in rats].

In a two-factorial balance trial two dietary protein levels of 12 and 15% the effect of a supplement of 50 mg alpha-glucosidase inhibitor (Bay g 5421) per kilogram food on nutrient digestibility, protein, and energy balance was tested in growing rats. At both protein levels the alpha-glucosidase inhibitor reduced the apparent digestibility of protein by maximal 8 and 4%, those of starch and energy only by 3 and 1%, respectively. With the low protein supply the protein and energy retention was decreased each by 13% when the inhibitor was given. On the basis of metabolizable energy, 6 and 19% less energy in form of protein and fat, respectively, have been deposited compared with the control group. These results may indicate also an effect of the glucosidase inhibitor on the intermediary metabolism.

Acarbose↗
PubMed1977

A comparative view on trace elements and growth.

The term 'essentiality' of trace elements is interpreted. The major human diseases due to trace element deficience (Fe, Cu, Zn, Cr) are briefly considered. In growing animals the first clinical deficiency symptom most often is a growth retardation. In order to diagnose a deficient situation before the manifestation of clinical symptoms, model studies with growing animals are presented to show criteria that respond sensitively even to suboptimum intake of trace elements (Zn, Cu, Fe, Ni). Activity measurements of enzymes and hormones are found suitable. During the phase of reproduction, which is closely associated with growth, the trace element concentration in milk may provide an additional diagnostic means. In conclusion, the problems of diagnosing the supply status are discussed. A possibility for determination of the trace element requirement is indicated.

Animals↗
PubMed1976

[Cu requirement of pregnant and lactating rats].

Trials with graded Cu supplementation were carried out to establish the minimum and optimum Cu demands of pregnant, lactating and non-pregnants rats. The rats were investigated for the rate of live weight gains of the dams and young rats, for the number of stillbirths and, in particular, for the Cu status in the liver, body and blood serum of the animals and for coeruloplasmin activity. All Cu values obtained from the organs investigated had reached a saturation level at 8 mug Cu/g diet with the exception of the values for body Cu found in the dams that were killed on the day of delivery. The same results were obtained, for the above criteria, with lactating rats. So, it may be concluded that pregnant and lactating rats receive an optimum supply of Cu when they are fed a starch + casein diet containing 8 ppm Cu. For non-pregnant female rats, the limits that might be regarded as adequate were even below the 8 ppm level of dietary Cu. The saturation level for Cu was also found to be dependent, in regard of the criteria investigated, on the physiological state of the animals. It was found that, on the day of parturition, the level of Cu concentration and coeruloplasmin activity in the serum of the dams and the Cu content in the liver of the new-born rats were the best indicators for the state of Cu supply to the dams. However, after a sucking period of 17 days, the level of total Cu content in the dams was found to respond very sensitively to changes in the Cu supply.

Animals↗
PubMed1976

[Cu concentration in the milk of rats receiving diets with varying Cu content].

2 trials were conducted with female rats to investigate in which way the level of dietary Cu influenced the level of Cu concentrations in rat milk. The trials were made to establish whether data on the Cu content of milk might be used as indication of the level and state of Cu supply to the animal. In the period of colostrum formation the Cu content of the milk was shown to be clearly dependent on the level of dietary Cu. In later periods of lactation Cu concentrations in the milk (from 2 mg of Cu per kg of diet) were found to be unaffected by the Cu content of the diet. Constant Cu concentrations of between 2.5 to 3.0 mg per kg of milk were noted in this period. It was only in cases of extreme Cu deficiency (0.5 mg/kg of diet) that soon after parturition the Cu values decreased to and remained at a very low level of 1 mg per kg of milk. Generally, colostrum contained 4-6 times more Cu than ordinary milk (in all groups).

Animal Nutritional Physiological Phenomena↗