Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I

Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin I(129-166) complex demonstrated that cardiac troponin I(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and (15)N transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1-80)/troponin I(129-166) or troponin I(1-167) suggested that troponin I residues 81-128 do not interact strongly with troponin C but likely serve to modulate the interaction of troponin I(129-166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin I(129-166) binding the cardiac troponin C/troponin I(1-80) complex correlate with partial opening of the cardiac troponin C regulatory domain previously demonstrated by distance measurements using fluorescence methodologies. Fluorescence emission from cardiac troponin C(F20W/N51C)(AEDANS) complexed to cardiac troponin I(1-80) was used to monitor binding of cardiac troponin I(129-166) to the regulatory domain of cardiac troponin C. The apparent K(d) for cardiac troponin I(129-166) binding to cardiac troponin C/troponin I(1-80) was 43.3 +/- 3.2 microM. After bisphosphorylation of cardiac troponin I(1-80) the apparent K(d) increased to 59.1 +/- 1.3 microM. Thus, phosphorylation of the cardiac-specific N-terminus of troponin I reduces the apparent binding affinity of the regulatory domain of cardiac troponin C for cardiac troponin I(129-166) and provides further evidence for beta-adrenergic modulation of troponin Ca(2+) sensitivity through a direct interaction between the cardiac-specific amino-terminus of troponin I and the cardiac troponin C regulatory domain.     

Pecam-1 is a modulator of stat family member phosphorylation and localization: lessons from a transgenic mouse

PECAM-1 (CD31) is a member of the immunoglobin (Ig) superfamily of cell adhesion molecules whose expression is restricted to hematopoietic and vascular cells. PECAM-1 can recruit adapter and signaling molecules via its immunoreceptor tyrosine activation motif (ITAM), suggesting that PECAM-1 plays a role in signal transduction pathways. To study the involvement of PECAM-1 in signaling cascades in vivo, we used the major histocompatibility (MHC) I gene promoter to target ectopic PECAM-1 expression in transgenic mice. We noted an attenuation of mammary gland development at early stages of virgin ductal branching morphogenesis. STAT5a, a modulator of milk protein gene expression during lactation, was localized to the nuclei of ductal epithelial cells of 6-week-old virgin PECAM-1 transgenics, but not in control mice. This correlated with decreases in ductal epithelial cell proliferation and induction of p21, an inhibitor of cell cycle progression. Using in vitro model systems we demonstrated PECAM-1/STAT5a association and found that residue Y701 in PECAM-1's cytoplasmic tail is important for PECAM-1/STAT5 association and that PECAM-1 modulates increases in STAT5a tyrosine phosphorylation levels. We suggest that by serving as a scaffolding, PECAM-1 can bring substrates (STAT5a) and enzymes (a kinase) into close proximity, thereby modulating phosphorylation levels of selected proteins, as previously noted for beta-catenin.     

Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.     

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.     

Use of a permanent magnetic field to inhibit the development of canine osteoarthritis

This study was designed to determine the potential of a permanent magnetic field to inhibit the progression of osteoarthritis (OA) in a canine model. The magnetic field was created by 72 domino-sized ceramic magnets with surface field strength of 1100 G (0.11 T). The magnetic field strength at the surface of the mattress was 450-500 G (45-50 mT) and was equally distributed over the mattress surface. Eighteen animals had closed resection of their right stifle anterior cruciate ligament. Their kennel floors were covered in one of three ways: no floor mattress (OA) (N = 6); a floor mattress with domino-sized ceramic pieces placed between two layers of foam (sham control OA-MAT) (N = 6); or a floor mattress with domino-sized ceramic permanent magnets placed between two layers of foam (OA-MAT-MAG) (N = 6). Animals were kept in their cages except for 4 h of exercise each day. The left stifle of six animals served as the normal control. The stifle joints were examined at 12 weeks for synovial effusion, gross anatomic appearance, microscopic anatomic appearance (Mankin score), and metalloproteinase (MMP)-1 and -3. Macroscopically, the OA-MAT-MAG group appeared to have less synovitis, less synovial effusion, less disruption of the cartilage surface, and less cartilage ulceration than did the OA group or the control mattress group. The mean Mankin score for the OA-MAT-MAG group was less than that for the OA group (4.2 +/- 0.8 vs. 6.7 +/- 0.3; P <.05) and the control mattress group (4.2 +/- 0.8 vs. 5.2 +/- 0.8; P >.05), but greater than that for the normal left group (4.2 +/- 0.8 vs. 1.0 +/- 0.4; P <.05). These scores show a trend of improvement for OA-MAT-MAG group but the difference with the sham control OA-MAT group was not statistically significant. In immunohistochemical studies, the OA-MAT-MAG group cartilage was stained less heavily for MMP-1 and MMP-3 than were the OA group cartilage and the control mattress group cartilage, but did not differ significantly in MMP-1 and MMP-3 from the normal left group cartilage. The OA-MAT-MAG group did not differ from the normal left group in MMP-3 as determined by Western blot analysis. The study suggests that OA of the medial femoral condyle developed in a canine model exposed to a magnetic field may be inhibited beyond the benefit provided by mattress. Further studies are needed to delineate more precisely the effect of the magnetic field in reducing the severity of OA.     

Brief inactivation of c-Myc is not sufficient for sustained regression of c-Myc-induced tumours of pancreatic islets and skin epidermis

Background  

Tumour regression observed in many conditional mouse models following oncogene inactivation provides the impetus to develop, and a platform to preclinically evaluate, novel therapeutics to inactivate specific oncogenes. Inactivating single oncogenes, such as c-Myc, can reverse even advanced tumours. Intriguingly, transient c-Myc inactivation proved sufficient for sustained osteosarcoma regression; the resulting osteocyte differentiation potentially explaining loss of c-Myc's oncogenic properties. But would this apply to other tumours?