Acute and long-term proteome changes induced by oxidative stress in the developing brain.

The developing mammalian brain experiences a period of rapid growth during which various otherwise innocuous environmental factors cause widespread apoptotic neuronal death. To gain insight into developmental events influenced by a premature exposure to high oxygen levels and identify proteins engaged in neurodegenerative and reparative processes, we analyzed mouse brain proteome changes at P7, P14 and P35 caused by an exposure to hyperoxia at P6. Changes detected in the brain proteome suggested that hyperoxia leads to oxidative stress and apoptotic neuronal death. These changes were consistent with results of histological and biochemical evaluation of the brains, which revealed widespread apoptotic neuronal death and increased levels of protein carbonyls. Furthermore, we detected changes in proteins involved in synaptic function, cell proliferation and formation of neuronal connections, suggesting interference of oxidative stress with these developmental events. These effects are age-dependent, as they did not occur in mice subjected to hyperoxia in adolescence.

Mimetics of a T cell epitope based on poly-N-acylated amine backbone structures induce T cells in vitro and in vivo.

Peptidomimetics of the major histocompatibility complex (MHC) class I-restricted ovalbumin-derived T cell epitope SIINFEKL were generated by replacing parts of the peptide backbone by a poly-N-acylated amine (PAA) backbone with aromatic, heteroaromatic, and pseudoaromatic side chains that branch off of the main chain at the amine nitrogen. The structure of the PAAs was designed to position this side chain in the central epitope anchor pocket of the MHC molecule. A number of biologically active PAAs were found that induced cytolysis by the mouse cytotoxic T cell clone 4G3. Competition experiments with independent peptides that are known to bind to the restricting MHC molecule H-2K(b) suggest that the PAAs are bound by the MHC molecules at the same site as conventional peptide epitopes. The PAAs were active also in vivo and induced primary cytotoxic T cell responses in mice.

Cutting edge: N-hydroxy peptides: a new class of TCR antagonists.

TCR antagonists are altered T cell epitopes that specifically inactivate T cells. Commonly, they are derived from agonists by amino acid side chain replacement at positions accessible to the TCR. In this paper we report for the first time that a main chain N-hydroxylation, which is not exposed at the surface of the MHC peptide complex, renders an agonist into an antagonist. These mimotopes are a new, yet undescribed class of TCR antagonists. The antagonists are about 100 times more potent than an unrelated peptide that competes for binding to the MHC molecule. The novel main chain modification enhances biostability and maintains side chain constitution and thus opens new prospects for the use of TCR antagonists in the treatment of pathological immune reactions.

New synthetic non-peptide ligands for classical major histocompatibility complex class I molecules.

Poly-N-acylated amines, as a new class of synthetic non-peptide ligands for the murine major histocompatibility complex (MHC) class I molecule H-2Kb, were developed on the basis of the ovalbumin-derived peptide epitope SIINFEKL. Non-peptidic structural elements were introduced at the C-terminal part of the ligand and include the two dominant anchors at positions 5 and 8. Several oligomers and five different combinatorial libraries were synthesized and tested for their H-2Kb-binding capacities in an MHC stabilization assay. First, the optimal spacing and geometry of the side chains were determined using a series of oligomers with different main chain modifications. Then, based on the structure with the highest binding efficiency, randomized libraries were designed that contain 26 different aromatic, heteroaromatic, or pseudoaromatic side chains for the dominant anchor at position 5, which is deeply buried inside the MHC peptide-binding groove and is crucial for the conformational stability of the entire peptide-MHC complex. Similarly, a series of aliphatic side chains were tested for the second dominant anchor at position 8. MHC-binding and MHC-stabilizing oligomers with defined structures were derived from these libraries by deconvolution.

N-Hydroxy-amide analogues of MHC-class I peptide ligands with nanomolar binding affinities.

A novel class of major histocompatibility complex class I (MHC-I) ligands containing an N-hydroxyamide bond was designed on the basis of the natural epitope SIINFEKL, and synthesized on solid phase. The capacity of these compounds to bind to the MHC-I molecule H-2Kb and to induce T cell responses was analysed in comparison with the corresponding glycine containing variant of SIINFEKL. Binding to the MHC molecule was diminished by the N-hydroxy group at positions 2 and 3 of the oligomer and improved in the case of positions 4, 5, 6 and 7. No change was seen for position 1. The efficacy of T cell stimulation was strongly reduced by the modification of all positions except for position 1. A complete loss of activity was found for the N-hydroxy variant in positions 4 and 6. N-Hydroxy amide-containing peptides displayed an enhanced stability to enzymatic degradation. This new class of MHC ligand can become instrumental as immunomodulatory reagent in various disease situations.

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation.

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.

Scent marking (pasting) in a colony of immature spotted hyenas (Crocuta crocuta): a developmental study.

Pasting, a stereotypic form of anal gland scent marking, was studied in 2 cohorts (N = 20) of captive spotted hyenas (Crocuta crocuta). A significant increment in the frequency of pasting occurred in both cohorts as the animals approached sexual maturity; however, gonadectomy during the early juvenile age period had no significant effects on subsequent pasting frequency. Dominant hyenas in both cohorts tended to scent-mark more frequently than subordinates during the late subadult period. Pasting was facilitated by the immediately preceding pasting activities of other hyenas, as has been reported to occur in nature, and olfactory investigation was the most common behavior preceding pasting.