Molecular determinants of sugar transport regulation by ATP

Intracellular ATP inhibits human erythrocyte net sugar transport by binding cooperatively to the glucose transport protein (GluT1). ATP binding produces altered transporter affinity for substrate and promotes substrate occlusion within a post-translocation vestibule formed by GluT1 cytosolic domains. The accompanying paper (Cloherty, E. K., Levine, K. B., Graybill, C., and Carruthers, A. (2002) Biochemistry 41, 12639-12651) demonstrates that reduced intracellular pH promotes high-affinity ATP binding to GluT1 but inhibits ATP-modulation of GluT1-mediated sugar transport. The present study explores the role of GluT1 residues 326-343 (a proposed GluT1 ATP-binding site subdomain) in GluT1 ATP binding by using alanine scanning mutagenesis. Cos-7 and HEK cells were transfected with a cDNA encoding full-length human GluT1 terminating in a carboxyl-terminal hemagglutinin (HA)-His6 epitope. The transporter (GluT1.HA.H6) is expressed at the surface of both cell-types and is catalytically active. In HEK cells, both parental GluT1- and GluT1.HA.H6-mediated sugar transport are acutely sensitive to cellular metabolic inhibition. Isolated, detergent-solubilized GluT1.HA.H6 is photolabeled by [gamma-32P]-azidoATP in an ATP-protectable manner. Alanine substitution of E329 or G332/R333/R334 enhances GluT1.HA.H6 [gamma-32P]azidoATP photoincorporation but blocks acute modulation of net sugar transport by cellular metabolic inhibition. These actions resemble those of reduced pH on ATP binding to and modulation of red cell GluT1. It is proposed that cooperative nucleotide binding to GluT1 and nucleotide modulation of GluT1-mediated sugar transport are regulated by a proton-sensitive saltbridge (Glu329-Arg333/334).     

Metacognition and reasoning

This article considers the cognitive architecture of human meta-reasoning: that is, metacognition concerning one's own reasoning and decision-making. The view we defend is that meta-reasoning is a cobbled-together skill comprising diverse self-management strategies acquired through individual and cultural learning. These approximate the monitoring-and-control functions of a postulated adaptive system for metacognition by recruiting mechanisms that were designed for quite other purposes.     

The discovery of potent selective NPY Y2 antagonists

A series of small molecules with a piperidinyl core were synthesized and tested for binding affinity (IC₅₀) at human Neuropeptide Y Y₂ receptor. Various amide related analogs (ureas, reversed amides, and sulfonamides) were evaluated. Several potent and selective NPY Y₂ antagonists were identified.     

Subunit molecular mass assignment of 14,654 Da to the soluble beta-galactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation.

The soluble dimeric beta-galactoside-binding lectin (subunit molecular mass, approximately 14 kDa) of bovine heart muscle, in common with the 14-kDa lectins of several other animal species, displays carbohydrate-binding activity when it is in the reduced state, but the purified lectin loses this activity upon oxidation. In the present study, the presence of any post-translational modification and the mechanism of the oxidative inactivation have been investigated by analyses of the reduced and oxidized forms of the purified bovine lectin by electrospray ionization-mass spectrometry (ESI-MS) and by liquid secondary ion mass spectrometry (LSIMS) of tryptic and peptic peptides. By ESI-MS, the molecular mass of the reduced lectin is determined to be 14,654.6 +/- 0.9 Da, and that of the oxidized lectin is 14,649.3 +/- 1.1 Da. These masses correspond to the amino acid sequence of the protein with the cysteines having free sulfhydryl groups in the reduced state and forming disulfide bonds in the oxidized state. There is no evidence of post-translational modification in either lectin form except for monoacetylation already predicted for alanine at the blocked N-terminal end. Pronounced differences in charge distribution in the electrospray ionization mass spectra of the reduced and oxidized lectin, reflecting a change in the number of accessible protonation sites in the oxidized protein, are consistent with the protein being held in an altered conformation by covalent bonding. The results of LSIMS analyses of tryptic and peptic peptides in conjunction with Edman sequencing indicate that disulfide bonding occurs predominantly between Cys2 and Cys130, Cys16 and Cys88, and Cys42 and Cys60. There is no evidence of oxidation of Trp68. These results, taken together with observations that almost the complete polypeptide chain is necessary for the functional integrity of the carbohydrate recognition domain (Abbott, W. M., and Feizi, T. (1991) J. Biol. Chem. 266, 5552-5557) point to intramolecular disulfide bonding with a change in protein folding and conformation as the mechanism of oxidative inactivation of the purified bovine lectin.     

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.     

Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.     

A Comprehensive Panel of Near-Full-Length Clones and Reference Sequences for Non-Subtype B Isolates of Human Immunodeficiency Virus Type 1

Non-subtype B viruses cause the vast majority of new human immunodeficiency virus type 1 (HIV-1) infections worldwide and are thus the major focus of international vaccine efforts. Although their geographic dissemination is carefully monitored, their immunogenic and biological properties remain largely unknown, in part because well-characterized virological reference reagents are lacking. In particular, full-length clones and sequences are rare, since subtype classification is frequently based on small PCR-derived viral fragments. There are only five proviral clones available for viruses other than subtype B, and these represent only 3 of the 10 proposed (group M) sequence subtypes. This lack of reference sequences also confounds the identification and analysis of mosaic (recombinant) genomes, which appear to be arising with increasing frequency in areas where multiple sequence subtypes cocirculate. To generate a more representative panel of non-subtype B reference reagents, we have cloned (by long PCR or lambda phage techniques) and sequenced 10 near-full-length HIV-1 genomes (lacking less than 80 bp of long terminal repeat sequences) from primary isolates collected at major epicenters of the global AIDS pandemic. Detailed phylogenetic analyses identified six that represented nonrecombinant members of HIV-1 subtypes A (92UG037.1), C (92BR025.8), D (84ZR085.1 and 94UG114.1), F (93BR020.1), and H (90CF056.1), the last two comprising the first full-length examples of these subtypes. Four others were found to be complex mosaics of subtypes A and C (92RW009.6), A and G (92NG083.2 and 92NG003.1), and B and F (93BR029.4), again emphasizing the impact of intersubtype recombination on global HIV-1 diversification. Although a number of clones had frameshift mutations or translational stop codons in major open reading frames, all the genomes contained a complete set of genes and three had intact genomic organizations without inactivating mutations. Reconstruction of one of these (94UG114.1) yielded replication-competent virus that grew to high titers in normal donor peripheral blood mononuclear cell cultures. This panel of non-subtype B reference genomes should prove valuable for structure-function studies of genetically diverse viral gene products, the generation of subtype-specific immunological reagents, and the production of DNA- and protein-based subunit vaccines directed against a broader spectrum of viruses.One critical question facing current AIDS vaccine development efforts is to what extent human immunodeficiency virus type 1 (HIV-1) genetic variation has to be considered in the design of candidate vaccines (11, 21, 41, 72). Phylogenetic analyses of globally circulating viral strains have identified two distinct groups of HIV-1 (M and O) (33, 45, 61, 62), and 10 sequence subtypes (A to J) have been proposed within the major group (M) (29, 30, 45, 72). Sequence variation among viruses belonging to these different lineages is extensive, with envelope amino acid sequence variation ranging from 24% between different subtypes to 47% between the two different groups. Given this extent of diversity, the question has been raised whether immunogens based on a single virus strain can be expected to elicit immune responses effective against a broad spectrum of viruses or whether vaccine preparations should include mixtures of genetically divergent antigens and/or be tailored toward locally circulating strains (11, 21, 41, 72). This is of particular concern in developing countries, where multiple subtypes of HIV-1 are known to cocirculate and where subtype B viruses (which have been the source of most current candidate vaccine preparations [10, 21]) are rare or nonexistent (5, 24, 40, 72).Although the extent of global HIV-1 variation is well defined, little is known about the biological consequences of this genetic diversity and its impact on cellular and humoral immune responses in the infected host. In particular, it remains unknown whether subtype-specific differences in virus biology exist that have to be considered for vaccine design. Thus far, such differences have not been identified. For example, several studies have shown that there is no correlation between HIV-1 genetic subtypes and neutralization serotypes (38, 42, 46, 68). Some viruses are readily neutralized, while most are relatively neutralization resistant (42). Although the reasons for these different susceptibilities remain unknown, it is clear that neutralization is not a function of the viral genotype (38, 42, 46, 68). Similarly, recent studies have identified vigorous cross-clade cytotoxic T-lymphocyte (CTL) reactivities in individuals infected with viruses from several different clades (3, 6), as well as in recipients of a clade B vaccine (15). These results are very encouraging, since they suggest that CTL cross-recognition among HIV-1 clades is much more prevalent than previously anticipated and that immunogens based on a limited number of variants may be able to elicit a broad CTL response (6). Nevertheless, it would be premature to conclude that HIV-1 variation poses no problem for AIDS vaccine design. Only a comprehensive analysis of genetically defined representatives of the various groups and subtypes will allow us to judge whether certain variants differ in fundamental viral properties and whether such differences will have to be incorporated into vaccine strategies. Obviously, such studies require well-characterized reference reagents, in particular full-length and replication-competent molecular clones that can be used for functional and biological studies.Full-length reference sequences representing the various subtypes are also urgently needed for phylogenetic comparisons. Recent analyses of subgenomic (23, 52, 54, 58) as well as full-length (7, 18, 53, 60) HIV-1 sequences identified a surprising number of HIV-1 strains which clustered in different subtypes in different parts of their genome. All of these originated from geographic regions where multiple subtypes cocirculated and are the results of coinfections with highly divergent viruses (52, 60, 62). Detailed phylogenetic characterization revealed that most of them have a complex genome structure with multiple points of crossover (7, 18, 53, 60). Some recombinants, like the “subtype E” viruses, which are in fact A/E recombinants (7, 18), have a widespread geographic dissemination and are responsible for much of the Asian HIV-1 epidemic (69, 70). In other areas, recombinants appear to be generated with increasing frequencies since many randomly chosen isolates exhibit evidence of mosaicism (4, 8, 31, 66, 71). Since recombination provides the opportunity for evolutionary leaps with genetic consequences that are far greater than those of the steady accumulation of individual mutations, the impact of recombination on viral properties must be monitored. We therefore need full-length nonrecombinant reference sequences for all major HIV-1 groups and subtypes before we can map and characterize the extent of intersubtype recombination.The number of molecular reagents for non-subtype B viruses is very limited. There are currently only five full-length, nonrecombinant molecular clones available for viruses other than subtype B (45), and these represent only three of the proposed (group M) subtypes (A, C, and D). Moreover, only three clones (all derived from subtype D viruses) are replication competent and thus useful for studies requiring functional gene products (45, 48, 65). Given the unknown impact of genetic variation on correlates of immune protection, subtype-specific reagents are critically needed for phylogenetic, immunological, and biological studies. In this paper, we report the cloning (by long PCR and lambda techniques) of 10 near-full-length HIV-1 genomes from isolates previously classified as non-subtype B viruses. Detailed phylogenetic analysis showed that six comprise nonmosaic representatives of five major subtypes, including two for which full-length representatives have not been reported. Four others were identified as complex intersubtype recombinants, again emphasizing the prevalence of hybrid genomes among globally circulating HIV-1 strains. We also describe a strategy for the biological evaluation of long-PCR-derived genomes and report the generation of a replication-competent provirus by this approach. The effect of these reagents on vaccine development is discussed.     

Distinct mechanisms govern proteolytic shedding of a key invasion protein in apicomplexan pathogens

Apical membrane antigen-1 (AMA1) is a conserved apicomplexan protein that plays an important but undefined role in host cell invasion. We have studied the fate of Plasmodium falciparum AMA1 (PfAMA1) during erythrocyte invasion by the malaria merozoite, and compared it with that of the Toxoplasma gondii orthologue, TgAMA1. Shedding of the PfAMA1 ectodomain goes essentially to completion during invasion, and occurs predominantly or exclusively via juxtamembrane cleavage at the previously identified sheddase cleavage site, Thr517. Only the resulting juxtamembrane stub of the ectodomain is efficiently carried into the host cell, and this remains distributed around the plasma membrane of the intracellular ring-stage parasite. Inhibition of normal shedding, however, results in proteolysis at an intramembrane, rhomboid-like cleavage site, and PfAMA1 is susceptible to cleavage by Drosophila rhomboid-1, showing that it can be a substrate for intramembrane cleavage but is not normally processed in this manner. In contrast, shedding of TgAMA1 from the surface of extracellular tachyzoites occurs exclusively via cleavage within the luminal half of its transmembrane domain by a rhomboid-like protease. Also unlike PfAMA1, complete TgAMA1 shedding does not accompany Toxoplasma invasion as the intact protein was readily detected on the surface of newly invaded tachyzoites. This work reveals unexpected differences in the manner in which Plasmodium and Toxoplasma shed AMA1 from the surface of invasive zoites, and demonstrates the presence at the malaria merozoite surface of a rhomboid-like protease.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.