Linear TMC-95-based proteasome inhibitors.

We have designed and evaluated 45 linear analogues of the natural constrained cyclopeptide TMC-95A. These synthetically less demanding molecules are based on the tripeptide sequence Y-N-W of TMC-95A. Structural variations in the amino acid side chains and termini greatly influenced both the efficiency and selectivity of action on a given type of active site.

Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein.

Signaling properties of anti-immunoglobulin--resistant variants of WEHI-231 B lymphoma cells.

Stimulation of the B cell antigen receptor (BCR) of the murine immature WEHI-231 B lymphoma with anti-immunoglobulin antibodies leads to irreversible growth arrest and apoptosis. As in normal B cells, membrane immunoglobulin (mIg) ligation in WEHI-231 cells triggers a series of signaling cascades from the BCR to intracellular compartments. In order to address the role of early signals in mediating the growth arrest of WEHI-231 cells, we have generated two variants resistant to the anti-Ig-mediated inhibitory effect.

Signaling of programmed cell death induction in WEHI-231 B lymphoma cells.

The murine WEHI-231 B lymphoma is highly sensitive to membrane immunoglobulin ligation which leads to programmed cell death (PCD) in this cell line. To study the molecular pathways involved in PCD induction in these cells, we derived two variants of WEHI-231 resistant to anti-Ig treatment. The level of bcl-2 mRNA was identical in the wild type and the variants, either untreated or anti-Ig treated, suggesting that PCD is not under the control of bcl-2 in WEHI-231 cells.

Expression and biological activity of interleukin-1 receptors in mouse gamma/delta thymocytes during ontogeny.

We have recently shown that the response of mouse thymocytes to interleukin (IL)-1 + IL-2 was maximal at birth and that the responding cells displayed a CD4-CD8- T cell receptor (TcR) gamma/delta + phenotype. Unexpectedly, despite their high proportion of gamma/delta + cells, fetal thymocyte populations responded only weakly to IL-1 + IL-2. In this report, we demonstrate that the discrepancy between the day 17.

Anti-immunoglobulins induce death by apoptosis in WEHI-231 B lymphoma cells.

Anti-membrane immunoglobulin (anti-mIg) antibodies exert inhibitory effects in immature B lymphocytes such as WEHI-231 cells. We show here that anti-mIg treatment causes DNA fragmentation (apoptosis) in these cells. We also report that co-treatment with the protein kinase C activator phorbol 12-myristate 13-acetate prevents apoptosis induced by anti-mIg.

Early membrane potential and cytoplasmic calcium changes during mitogenic stimulation of WEHI 231 cell line by polyene antibiotics, lipopolysaccharide and anti-immunoglobulin.

Changes in free cytosolic calcium concentration and in membrane voltage are thought to be important initiating events in lymphocyte activation. The antifungal agent amphotericin B (AmB) holds interesting immunomodulating properties and its N-thiopropionyl derivative (AmBSH) is a potent polyclonal B-cell activator. These molecules may then exert their stimulating activity through the production of early ionic signals similar to those delivered by the classical activators lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig).

Membrane effects of the polyene antibiotic amphotericin B and of some of its derivatives on lymphocytes.

Amphotericin B (AmB) exhibits immunomodulating properties in mice. In vitro studies on lymphocytes, in relation with these properties, are reported here with AmB and two of its derivatives: the N-Fructosyl (N-Fru AmB) and the N-thiopropionyl (AmBSH) derivatives. Interactions of these molecules with thymocytes, a sensitive cell type, demonstrated that the extent of binding is not a toxicity parameter.

Membrane IgM cross-linking is not coupled to protein kinase C translocation in WEHI-231 B lymphoma cells.

The early molecular events involved in the process of signal transduction via membrane immunoglobulins (mIg) include phosphatidyl inositol metabolism, intracellular Ca2+ mobilization, and protein kinase C (PKC) activation. Anti-mIg antibodies exert either stimulating or inhibitory effects depending on the activation state and/or the differentiation stage of B cells. WEHI-231 is a murine B lymphoma that becomes inactivated upon anti-mIg treatment.

Phorbol ester induces class II gene expression in pre-B cell lines.

The effect of phorbol myristate acetate on the induction of major histocompatibility complex class II gene expression was studied in four Abelson murine leukemia virus-transformed pre-B cell lines. In three cell lines, low concentrations of PMA (0.1-10 ng/ml) induced the expression of high levels of surface Ia molecules, and this effect was mediated at the transcriptional level.

Cyclic AMP-modulated potassium channels in murine B cells and their precursors.

A voltage-dependent potassium current (the delayed rectifier) has been found in murine B cells and their precursors with the whole-cell patch-clamp technique. The type of channel involved in the generation of this current appears to be present throughout all stages of pre-B-cell differentiation, since it is detected in pre-B cell lines infected with Abelson murine leukemia virus; these cell lines represent various phases of B-cell development. Thus, the presence of this channel is not obviously correlated with B-cell differentiation.

B cell triggering properties of a nontoxic derivative of amphotericin B.

The immunomodulating properties of amphotericin B (AMB), an antifungal polyene antibiotic, have been reported in multiple studies. However, many findings on the subject are conflicting, and the precise mechanism of AMB action on the immune system is yet unknown. Because toxicity and limited solubility of AMB are likely to be responsible for these discrepancies, we synthesized a nontoxic derivative of AMB (AMBSH), and we investigated its immune modulating effects on murine B cells.

Transport and hydrolysis of peptides in Saccharomyces cerevisiae.

The transport and hydrolysis of several radioactive di- and tripeptides in Saccharomyces cerevisiae was studied. A peptide-transport-deficient mutant isolated on the basis of its resistance to nikkomycin Z lost most of its capacity to take up di- and tripeptides. The transport kinetics of [14C]methionylglycine, [14C]methionylsarcosine and [3H]nikkomycin Z indicated that peptide transport is not dependent on intracellular hydrolysis.

Sensitivity to nikkomycin Z in Candida albicans: role of peptide permeases.

The uptake of tritiated nikkomycin Z, a potent inhibitor of chitin synthetase, is mediated by a peptide transport system in Candida albicans. Kinetic transport assays with radioactive di- and tripeptides and competition studies suggest that two distinct systems operate in this yeast. Nikkomycin Z was transported through one of these systems, common to di- and tripeptides.

Photoaffinity inhibition of peptide transport in yeast.

A photoaffinity label, 4-azidobenzoyltrimethionine has been synthesized. It competitively inhibits trimethionine uptake in the yeast C. albicans.