Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA-transfected Epstein-Barr virus-immortalized autologous B cells.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP3) binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) ≈2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 μM and Hill coefficient (Hinh) ≈4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.

Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL.

The Escherichia coli chaperonins GroEL and GroES facilitate the refolding of polypeptide chains in an ATP hydrolysis-dependent reaction. The elementary steps in the binding and release of polypeptide substrates to GroEL were investigated in surface plasmon resonance studies to measure the rates of binding and dissociation of a normative variant of subtilisin. The rate constants determined for GroEL association with and dissociation from this variant yielded a micromolar dissociation constant, in agreement with independent calorimetric estimates. The rate of GroEL dissociation from the nonnative chain was increased significantly in the presence of 5'-adenylylimidodiphosphate (AMP-PNP), ADP, and ATP, yielding maximal values between 0.04 and 0.22 s(-1). The sigmoidal dependence of the dissociation rate on the concentration of AMP-PNP and ADP indicated that polypeptide dissociation is limited by a concerted conformational change that occurs after nucleotide binding. The dependence of the rate of release on ATP exhibited two sigmoidal transitions attributable to nucleotide binding to the distal and proximal toroid of a GroEL-polypeptide chain complex. The addition of GroES resulted in a marked increase in the rate of nonnative polypeptide release from GroEL, indicating that the cochaperonin binds more rapidly than the dissociation of polypeptides. These data demonstrate the importance of nucleotide binding-promoted concerted conformational changes for the release of chains from GroEL, which correlate with the sigmoidal hydrolysis of ATP by the chaperonin. The implications of these findings are discussed in terms of a working hypothesis for a single cycle of chaperonin action.