Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7

Implementation of a Pharmacist-Directed Cardiovascular Risk and Medication Management Program for Participants in a Construction Trade Benefit Trust Fund

Objectives: (1) To report the results of a pharmacist-directed cardiovascular risk management program; and (2) to identify obstacles faced by the pharmacists in the program implementation. Methods: The collaborators in this study included two local unions, a health benefit consulting company, and a community pharmacy. A total of 750 union workers with cardiovascular risk were informed about the cardiovascular risk management program. The program lasted six months, and the participation was voluntary. There were three group educational sessions with each session followed by a medication management service. A staff person of the health benefit consulting company and two pharmacists were interviewed via telephone. The interview questions were created according to the Gaps Model of Service Quality. The Gaps Model theorizes five gaps among consumer expectations, consumer perceptions, management perceptions of consumer expectations, service quality, service delivery, and external communications to consumers. The following data were collected: (1) types and quantity of drug therapy problems, (2) pharmacists' recommendations and prescribers' response, (3) patients' quality of life, disability days, and sick days, and (4) the experience of involved parties. Descriptive statistics were calculated. Results: Fifteen union workers participated in the program. For the participants, 35 drug-related problems were identified, with "need for additional therapy" and "dose too low" being the most common problems. To address these drug-related problems, pharmacists made 33 recommendations to prescribers, and prescribers accepted 55% of the recommendations. According to the interviews, there were three barriers faced by pharmacists to implement the program: lack of consensus about the recruitment, union workers' unawareness of the program's benefits, and limited support from the unions and the health benefit consulting company. Conclusions: It was difficult to recruit participants into the program. Clear agreement among collaborators on both the program's benefits and the specific roles of each collaborator may be the key to successfully implement similar programs in the future. Type: Case Stud

etramps, a New Plasmodium falciparum Gene Family Coding for Developmentally Regulated and Highly Charged Membrane Proteins Located at the Parasite–Host Cell Interface

After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole and develops from morphologically and metabolically distinct ring to trophozoite stages. During these developmental phases, major structural changes occur within the erythrocyte, but neither the molecular events governing this development nor the molecular composition of the parasitophorous vacuole membrane (PVM) is well known. Herein, we describe a new family of highly cationic proteins from P. falciparum termed early transcribed membrane proteins (ETRAMPs). Thirteen members were identified sharing a conserved structure, of which six were found only during ring stages as judged from Northern and Western analysis. Other members showed different stage-specific expression patterns. Furthermore, ETRAMPs were associated with the membrane fractions in Western blots, and colocalization and selective permeabilization studies demonstrated that ETRAMPs were located in the PVM. This was confirmed by immunoelectron microscopy where the PVM and tubovesicular extensions of the PVM were labeled. Early expressed ETRAMPs clearly defined separate PVM domains compared with the negatively charged integral PVM protein EXP-1, suggesting functionally different domains in the PVM with an oppositely charged surface coat. We also show that the dynamic change of ETRAMP composition in the PVM coincides with the morphological changes during development. The P. falciparum PVM is an important structure for parasite survival, and its analysis might provide better understanding of the requirements of intracellular parasites