Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor-effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity

African American mothers living with HIV/AIDS : disclosure to an adolescent child, stigma, psychological distress and illness severity

Utilizing pre-existing quantitative and qualitative data from a sample of twenty-seven African American mother/adolescent dyads this study examined the process of maternal HIV disclosure as well as mother/adolescent communication regarding mothers\u27 illness and its relationship to mothers\u27 experience of HIV related stigma, psychological distress, illness severity and mother/adolescent reports of child functioning. Post-study interviews were conducted with forty percent of the mothers (N=10) who participated in the primary study to examine how mothers\u27 perceive the process of maternal HIV disclosure over time. Quantitative results demonstrated moderate to modest correlations between mothers\u27 experience of HIV related stigma, psychological distress, mothers\u27 perceptions of adolescents\u27 problem behaviors and mothers\u27 perceptions of ongoing HIV communication with their adolescents. Qualitative results revealed that the initial disclosure event is an affective and concrete process related to: (a) mothers\u27 experience prior to disclosure; (b) motivation for disclosure; (c) disclosure information; (d) mother\u27s perception of teens\u27 reaction to disclosure, and (e) mothers\u27 experience after disclosure. Post-study results highlight how the process of maternal HIV disclosure evolves overtime, and the persistent nature and influence of HIV related stigma, specifically perceived stigma, in mothers\u27 requests during the initial disclosure event and over time that their diagnosis be kept secret

Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites

The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites

An Exploratory Study of Students’ Perceptions of Environmental Issues as Social Work Practice and Their Understanding of Environmental Justice

As environmental crises continue to rise, the profession of social work in the US is being called to incorporate environmental justice content in the training and education of social workers to prepare them for addressing the effects of environmental injustices; however, students’ awareness of the profession’s environmental justice mission is uncertain. The current mixed-method study sought to explore this issue amongst a sample of social work students (N = 724) in the US. Overall, students mildly to moderately believe that environmental issues are part of practice, and regression results revealed that greater support was associated with the belief in climate change and completing a university course that included environmental injustice. Content analysis of an open-ended question that asked students to define ‘environmental justice’ revealed that students primarily defined this term within the context of general environmental harm (28%) and 15% linked this term to a disproportionate exposure to hazards among people who have been historically oppressed and marginalized. Together these results suggested that further educational efforts may be needed to expand students’ knowledge of environmental (in)justice and how it is part of practice across the micro-macro continuum

Are Social Work Students Concerned About the Environment?: The Role of Personal Beliefs

The profession of social work is committed to social justice issues, and part of this mission includes the environment. Specifically, the Council on Social Work Education supports equality in environmental justice, ecological unity among species, and using ecological resources in a responsible way. However, a dearth exists in the literature with regard to social work students’ concern about the environment and what predicts their support. This study sought to redress this gap. Social work students (N = 724) from programs throughout the country were surveyed about their environmental beliefs and behaviors. An ordinary least squares regression indicated that holding more liberal political beliefs, identifying as a nonreligious individual, placing greater personal importance on environmental issues, participating in environmentally conscious behaviors, espousing greater confidence in scientists’ understanding of climate change, and being older were associated with greater environmental concern, and these variables explained 51.5% of the variance. Greater inclusion of environmental justice and avenues for advocacy that create social change should be part of the social work curricula if practitioners who are ready for this area of practice across the micro–macro continuum are to be developed

Differential roles of the dopamine 1-class receptors, D1R and D5R, in hippocampal dependent memory

Pharmacological and global KO studies have worked to elucidate the function of dopamine 1-class receptors (D1Rs and D5Rs) in hippocampal-dependent learning and memory. Yet, these manipulations are unable to restrict D1R from D5R activity within hippocampal subregions. We generated mice that lack D1Rs or D5Rs in dentate gyrus (DG) granule cells of the hippocampus. This allowed us to characterize the precise role of D1Rs and D5Rs in modulating c-Fos activity in the hippocampus and in Pavlovian fear conditioning. We demonstrate that DG D1R deletion, but not D5R deletion, increases DG granule cell baseline c-Fos activity, decreases DG and CA3 c-Fos activity in response to contextual exposure and to contextual fear conditioning, impairs contextual memory formation, and enhances generalization of the conditioned fear response.Howard Hughes Medical InstituteRIKEN Brain Science Institut

Properties of the human erythrocyte glucose transport protein are determined by cellular context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific