Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

Three-dimensional structure of transporter associated with antigen processing (TAP) obtained by single Particle image analysis.

The transporter associated with antigen processing (TAP) is an ATP binding cassette transporter responsible for peptide translocation into the lumen of the endoplasmic reticulum for assembly with major histocompatibility complex class I molecules. Immunoaffinity-purified TAP particles comprising TAP1 and TAP2 polypeptides, and TAP2 particles alone were characterized after detergent solubilization and studied by electron microscopy. Projection structures of TAP1+2 particles reveal a molecule approximately 10 nm across with a deeply staining central region, whereas TAP2 molecules are smaller in projection. A three-dimensional structure of TAP reveals it is isolated as a single heterodimeric complex, with the TAP1 and TAP2 subunits combining to create a central 3-nm-diameter pocket on the predicted endoplasmic reticulum-lumenal side. Its structural similarity to other ABC transporters demonstrates a common tertiary structure for this diverse family of membrane proteins.

Early clinical manifestations of cannabis dependence in a community sample.

BACKGROUND: In this research, the 'natural history' of cannabis dependence is probed, using data from a large epidemiological sample of cannabis users followed from 1981 through 1996, until most of these users had passed through the empirically derived period of risk for developing cannabis dependence. METHODS: The Baltimore Epidemiologic Catchment Area research group sampled, recruited, and assessed 3481 adults age 18+ years in 1981. Survivors were re-assessed roughly 13 years later. Among 599 cannabis users, a total of 37 had become cases of cannabis dependence during the follow-up interval; 41 had developed DSM-IIIR cannabis abuse without dependence; 521 had suffered insufficient problems to qualify as cases. Survival analysis methods were used to plot and study the cumulative occurrence of these problems in a primary contrast of the 37 dependence cases and 562 non-cases. RESULTS: Subjectively felt loss of control over cannabis and continued cannabis use despite knowledge of harm seem to appear most rapidly among cases of cannabis dependence. In contrast, subjectively felt withdrawal symptoms tended to emerge later and for a much smaller proportion of dependence cases and non-cases. CONCLUSION: The 5000+ person-years of follow-up experience provided by the cannabis users in this study give an unprecedented look at the natural history and clinical course of cannabis dependence. Distinctive features of early cannabis dependence may help differentiate cannabis users who progress to clinically significant dependence from those who remain non-dependent.

Aggressive behavior and opportunities to purchase drugs.

Robins, Kellam, and others found robust evidence linking youthful aggression and deviance to later illicit drug use. Some investigators favor the interpretation that drug use is just one manifestation or complication of a more general problem behavior syndrome or conduct disorder. In this work, we test the complementary hypothesis that aggressive youth are more likely to be approached with offers to buy drugs, and found the most aggressive youths were about five times more likely to be offered drugs for purchase. However, this association was much attenuated when levels of delinquency were taken into account. In this respect, delinquent rather than aggressive behavior might be more salient. This study's evidence does not contradict previous problem behavior theories, but rather prompts new ideas about how aggression, delinquency, and drug use might be linked. One testable hypothesis is that youths with both aggression and delinquency are more likely to enter microenvironments where drug dealing is more prevalent. Or, their observable behaviors or physical appearance might function as signs of apparent willingness to try drugs. These results add to our understanding of links between aggression, delinquency, and drug use, and introduce a new line of epidemiological inquiry focused upon drug purchase opportunities.

The structure of the multidrug resistance protein 1 (MRP1/ABCC1). crystallization and single-particle analysis.

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH(2) terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are approximately 80 x 100 A. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to approximately 22-A resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.

Three-dimensional structure of herpes simplex virus type 1 glycoprotein D at 2.4-nanometer resolution.

Herpes simplex virus type 1 glycoprotein D (gD) is essential for virus infectivity and is responsible for binding to cellular membrane proteins and subsequently promoting fusion between the virus envelope and the cell. No structural data are available for gD or for any other herpesvirus envelope protein. Here we present a three-dimensional model for the baculovirus-expressed truncated protein gD1(306t) based on electron microscopic data. We demonstrate that gD1(306t) appears as a homotetramer containing a pronounced pocket in the center of the molecule. Monoclonal antibody binding demonstrates that the molecule is oriented such that the pocket protrudes away from the virus envelope.

Structure of the multidrug resistance P-glycoprotein.

In order to elucidate the mechanism by which the multidrug resistance P-glycoprotein extrudes cytotoxic drugs from the cell, and particularly the number and nature of the drug binding site(s), knowledge of the structure of P-gp is essential. A considerable body of genetic and biochemical data has accrued which gives insights into P-gp structure and function. These data are critically reviewed, particularly in relation to the low resolution structure of P-gp which has recently been determined by electron microscopy. P-gp is one of the best characterised of the ABC transporters and these structure-function studies may have more general implications.

Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis.

P-glycoprotein (P-gp) is a member of the ATP binding cassette superfamily of active transporters and can confer multidrug resistance on cells and tumors by pumping chemotherapeutic drugs from the cytoplasm. P-gp was purified from CHrB30 cells and retained the ability to bind substrates and hydrolyze ATP. Labeling of P-gp with lectin-gold particles suggested it is monomeric. An initial structure of purified P-gp was determined to 2.5 nm resolution by electron microscopy and single particle image analysis of both detergent-solubilized and lipid-reconstituted protein. The structure was further refined by three dimensional reconstructions from single particle images and by Fourier projection maps of small two-dimensional crystalline arrays (unit cell parameters: a, 14.2 nm; b, 18.5 nm; and gamma, 91.6 degrees ). When viewed from above the membrane plane the protein is toroidal, with 6-fold symmetry and a diameter of about 10 nm. There is a large central pore of about 5 nm in diameter, which is closed at the inner (cytoplasmic) face of the membrane, forming an aqueous chamber within the membrane. An opening from this chamber to the lipid phase is present. The projection of the protein perpendicular to the membrane is roughly rectangular with a maximum depth of 8 nm and two 3-nm lobes exposed at the cytoplasmic face of the membrane, likely to correspond to the nucleotide binding domains. This study provides the first experimental insight into the three-dimensional architecture of any ATP binding cassette transporter.

Structure of photosystem II in spinach thylakoid membranes: comparison of detergent-solubilized and native complexes by electron microscopy.

1. Electron microscopy of solubilized photosystem II (PSII) complexes and PSII in spinach thylakoid membranes has been carried out and the results have been compared with data obtained from ordered two-dimensional arrays of PSII. Membrane-bound PSII is roughly rectangular (17.6 nm x 14.1 nm) with a central stain cavity surrounded by four major lumenal domains. A comparison between the averaged projections of single (non-ordered) particles at 3.8 nm resolution and the Fourier projection maps obtained from ordered arrays (at 2-3 nm resolution) reveals close similarity and excludes the possibility that PSII observed in two-dimensional ordered arrays represents an unusual subpopulation. 2. After detergent solubilization, PSII adopts various aggregation states which were analysed by electron microscopy in conjunction with single-particle averaging. Two different types of projection of roughly rectangular shape and of dimensions 30 nm x 17 nm manifesting themselves as tetrameric sandwich structures have been revealed. This conclusion is supported by the presence of at least two axes of 2-fold rotational symmetry running perpendicular to each other and intersecting at the centre of the oligomer. Comparisons of the structures of detergent-solubilized and native PSII show that the oligomerization of PSII can be artificially induced by the process of membrane solubilization.

A liposomal enzyme electrode for measuring glucose.

Enzyme electrodes have been described for measuring glucose but have been limited by the saturation kinetics of the glucose oxidase not allowing clinically relevant glucose concentrations to be measured (0-25 mM). One way of alleviating this problem is to use diffusion-controlled membranes which result in the enzyme experiencing a smaller substrate concentration than that of the bulk solution. As an extension of this concept we have encapsulated glucose oxidase in liposomes whereby the lipid bilayer wall provides the diffusion-limiting membrane as well as providing a biocompatible layer which is of particular relevance when blood glucose is to be measured. Linear ranges were found to embrace the required glucose concentrations and moreover by using liposomes prepared from different lipids, e.g., dimyristoyl (14:0) phosphatidylcholine (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC) and distearoyl (18:0) phosphatidylcholine (DSPC), the electrode response was shown to depend on the bilayer permeabilities in relation to the lipid phase transition temperatures and as a consequence the linear ranges were duly altered.