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.     

Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants "mat" for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.     

A comparison of isoload and isoinertial leg press training on bone and muscle outcomes

The subjects of this study (n = 20; 16 women, 4 men) performed 10 weeks of leg press training using one of two exercise modes (isoload or isotonic) with no crossover. Their workouts, which were performed 3 times per week, involved 4 sets of 8 repetitions with maximal voluntary effort. Testing was performed pre- and posttraining to examine bone and muscle changes. Posttraining, both groups incurred significant concentric knee extensor strength and leg muscle mass gains, while the percentage of body fat and total body fat mass each decreased. Leg and total body bone mineral densities showed group-by-time interactions, as isoload exercise caused posttraining gains, while isoinertial values were unchanged. Bone resorption assays showed insignificant changes. Isoload training likely involved greater strain magnitudes and rates to evoke higher peak forces and osteogenesis. Transduction of the training stimulus may have involved (a) formation in response to microdamage, and (b) piezoelectric-induced potentials that stimulated site-specific osteoblast activity and osteogenesis.     

Frontal plane moments do not accurately reflect ankle dynamics during running

The ankle joint has typically been treated as a universal joint with moments calculated about orthogonal axes and the frontal plane moment generally used to represent the net muscle action about the subtalar joint. However, this joint acts about an oblique axis. The purpose of this study was to examine the differences between joint moments calculated about the orthogonal frontal plane axis and an estimated subtalar joint axis. Three-dimensional data were collected on 10 participants running at 3.6 m/s. Joint moments, power, and work were calculated about the orthogonal frontal plane axis of the foot and about an oblique axis representing the subtalar joint. Selected parameters were compared with a paired t-test (alpha = 0.05). The results indicated that the joint moments calculated about the two axes were characteristically different. A moment calculated about an orthogonal frontal plane axis of the foot resulted in a joint moment that was invertor in nature during the first half of stance, but evertor during the second half of stance. The subtalar joint axis moment, however, was invertor during most of the stance. These two patterns may result in qualitatively different interpretations of the muscular contributions at the ankle during the stance phase of running.     

TRPC1 forms the stretch-activated cation channel in vertebrate cells

The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na(+), K(+), Ca(2+) and Mg(2+)) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa (>or=10(7)MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (M(r) 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.     

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).     

Vegetation regeneration on blanket mire after mechanized peat-cutting

• 1 Blanket mire in Northern Ireland is an ecologically threatened habitat in which land use for hand peat‐cutting, forestry and agriculture has had a major influence. A recent land use change is the introduction of tractor‐powered peat‐harvesting. In this paper, the effect of machine peat‐cutting on ombrotrophic blanket mire vegetation is assessed from a regional sample of cut and uncut plots.
• 2 Principal components analysis identified water‐table depth and grazing intensity as major factors influencing the species composition of uncut mire. A key variable affecting the composition of machine‐cut mire across the drainage gradient was the number of times cut, with multiple annual cutting causing progressive decreases in acrotelm depth, catotelm bulk density and plant cover. Ericaceous species and Sphagnum spp. were particularly sensitive to cutting, with Eriophorum angustifolium and Campylopus introflexus characteristic of multiple‐cut sites.
• 3 Redundancy analysis, with number of times cut partialled out, showed that recovery time accounted for a significant amount of variance in vegetation composition. Species that significantly increased in abundance with recovery time were Sphagnum spp., Odontoschisma sphagni , Erica tetralix and Drosera rotundifolia.
• 4 Sites cut frequently, or which were grazed, recovered more slowly. Recovery from cutting was partly dependent on the post‐cutting structure of the mire surface and the species that survive cutting. The rate of recovery on sites cut once, then abandoned, is relatively rapid compared with multiple‐cut sites where species colonization is constrained by bare compacted peat.

     

Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling?

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.