NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

An Innovative Family Preservation Program in an African American Community: Longitudinal Analysis

This paper presents a secondary analysis of data from a longitudinal evaluation of a community-based family preservation program in Portland, Oregon, designed for and by African Americans. Families served by the Family Enhancement Program (FEP) resemble chronically neglecting families in terms of numbers of children and length of contact with child protective services. Six- and twelve-month follow-ups for FEP clients were compared to data on families served by the Oregon State Office of Services to Children and Families (SOSCF). The author found that FEP families are more likely than SOSCFfamilies to show greater improvement between the pretest scores and the posttest scores for number of days in placement, number of placements, and number of founded maltreatment reports.

Inputs to parasol ganglion cells in the primate retina

Retinal ganglion cells carry signals from the eye to the brain. One of the most common types of ganglion cells is parasol cells. They have larger dendritic trees, somas and axons than other ganglion cells. While much was known about parasol cell light responses, little was known about how these responses are formed. One possibility is that they receive input from a unique set of local circuit neurons that have similar responses. The goal was to identify these presynaptic neurons and study their synaptic connectivity.^ Ganglion cells receive input from bipolar and amacrine cells, but there are numerous subtypes of each. To determine which of these were most likely to provide input to parasol cells, the parasol cells were intracellularly-injected and then various bipolar and amacrine cells were immunolabeled and the tissue analyzed using a confocal microscope. DB3 bipolar cells labeled with antibodies to calbindin made extensive contacts with OFF parasol cells. Antibodies to recover in labeled flat midget bipolar cells (FMB). They made only random contacts with OFF parasol cells, and they are not expected to provide significant input. Type DB2 bipolar cells and FMB cells labeled with antibodies to excitatory amino acid transporter-2 made extensive contacts with OFF parasol cells. This suggests that DB2 bipolar cells are likely to provide input to parasol cells.^ Two types of amacrine cells were labeled in material containing injected parasol cells. Cholinergic amacrine cells were labeled with antibodies to choline acetyltransferase, and they made extensive contacts with ON parasol cells. The large amacrine cells labeled with antibodies to a precursor of cholecystokinin were among the amacrine cells that are tracer-coupled to parasol cells.^ From electron microscopic (EM) analysis, most of the synapses made by DB3 axons were found on varicosities. Some postsynaptic and presynaptic amacrine cells resembled AII amacrine cells. Others were relatively electron-lucent and may be cholinergic amacrine cells or cholecystokinin-containing amacrine cells. Gap junctions were found between neighboring DB3 axons. They occurred whenever two axons contacted each other, and the junctions were as large as the area of contact. In double-label EM experiments, DB3 axons made synapses onto OFF parasol cells. ^

Community health workers: The critical link to sustainable, healthy societies in low-resource settings---A model for the Baylor Shoulder to Shoulder Foundation

Community health workers (CHWs) can serve as a bridge between healthcare providers and communities to positively impact social determinants of health and, thus, the overall health of the population. The potential to effect lasting change is particularly significant within resource-poor settings with limited access to formally trained health care providers such as the small, rural village of Santa Ana Intibucá, Honduras and surrounding areas—located on the geographically and politically isolated border of Honduras and El Salvador. The Baylor Shoulder to Shoulder Foundation (BSTS) works in conjunction with Santa Ana's volunteer health committee to bring a health brigade that has provided health care and public health projects to the area at least twice a year since 2001. They have also hired a full-time Honduran physician, a Honduran in-country administrative director, and built a clinic; yet, no community health worker program exists. This CHW program model is the response to a clear need for a CHW program within the area served by BSTS and presents a CHW program model specific to Santa Ana Intibucá and surrounding areas to be implemented by BSTS. Methods used to develop this model include reviewing the literature for recommendations from leading authorities as well as successfully implemented CHW programs in comparable regions. This information was incorporated into existing knowledge and materials currently being used in the area. Using the CHW model proposed here, each brigade, in conjunction with the communities served, can help develop new modules to respond to the specific health priorities of the region at that time, incorporating consistent modes of contact with the local physician and the CHWs to provide refresher courses, training in new topics of interest, and to be reminded of the importance of community health workers' role as the critical link to healthy societies. With cooperation, effort, and support, the brigade can continue to help integrate a sustainable CHW system in which communities may be able to maximize the care they receive while also learning to care for their own health and the future of their communities.^