A direct test of the reductionist approach to structural studies of calmodulin activity: relevance of peptide models of target proteins

Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM.CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM.CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM.CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme.     

Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2(b), IE(-)) mice were given 10(8) spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2(d), IE(+)) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 10(7) T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vbeta11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.     

Manipulation of dendritic cells as an approach to improved outcomes in transplantation

At the time of organ transplantation, a variety of non-parenchymal cells are transplanted simultaneously with the allograft. Recognition of the importance of these cells as potential immunostimulatory cells lead to the concept of 'passenger leukocytes' as the principal instigators of rejection. Passenger leukocytes include interstitial dendritic cells (DCs) and blood-derived monocytes/macrophages. As investigators have discovered the significance of DCs in influencing graft outcome, so have they begun to determine the best ways to influence DCs themselves. This review discusses the role of DCs in transplantation and then focuses on three different approaches for manipulating DCs to improve allograft survival: (1) targeting of chemokines involved in DC migration, (2) pharmacological arrest of DC maturation, and (3) genetic engineering of DCs.     

Immunotherapeutic potential of whole tumour cells

Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.     

Crystal structure of a tetradecameric assembly of the association domain of Ca2+/calmodulin-dependent kinase II

We report the crystal structure of the 143 residue association domain of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The association domain forms a hub-like assembly, composed of two rings of seven protomers each, which are stacked head to head and held together by extensive interfaces. The tetradecameric organization of the assembly was confirmed by analytical ultracentrifugation and multiangle light scattering. Individual protomers form wedge-shaped structures from which N-terminal helical segments that connect to the kinase domain extend toward the equatorial plane of the assembly, consistent with the arrangement of the kinase domains in a second outer ring. A deep and highly conserved pocket present within the association domain may serve as a docking site for proteins that interact with CaMKII.     

Glucose kinetics and exercise performance during phases of the menstrual cycle: effect of glucose ingestion

To study the effect of menstrual cycle phase and carbohydrate ingestion on glucose kinetics and exercise performance, eight healthy, moderately trained, eumenorrheic women cycled at 70% of peak O(2) consumption for 2 h and then performed a 4 kJ/kg body wt time trial. A control (C) and a glucose ingestion (G) trial were completed during the follicular (F) and luteal (L) phases of the menstrual cycle. Plasma substrate concentrations were similar before the commencement of exercise. Glucose rates of appearance and disappearance were higher (P < 0.05) during the 2nd h of exercise in FC than in LC. The percent contribution of carbohydrate to total energy expenditure was greater in FC than in LC, and subjects performed better (13%, P < 0.05) in FC. Performance improved (19% and 26% in FG and LG compared with FC and LC, respectively, P < 0.05) with the ingestion of glucose throughout exercise. These data demonstrate that substrate metabolism and exercise performance are influenced by the menstrual cycle phase, but ingestion of glucose minimizes these effects.