Adenosine Stimulates Cone Photoreceptor Myoid Elongation via an Adenosine A2-Like Receptor

In several parts of the nervous system, adenosine has been shown to function as an extracellular neuromodulator binding to surface receptors on target cells. This study examines the possible role of adenosine in mediating light and circadian regulation of retinomotor movements in teleost cone photoreceptors. Teleost cones elongate in the dark and contract in the light. In continuous darkness, the cones continue to elongate and contract at subjective dusk and dawn in response to circadian signals. We report here that exogenous adenosine triggers elongation (the dark/night movement) in isolated cone inner segment-cone outer segment preparations (CIS-COS) in vitro. Agonist/antagonist potency profiles indicate that adenosine's effect on cone movement is mediated by an A2-like adenosine receptor, which like other A2 receptors enhances adenylate cyclase activity. Although closest to that expected for A2 receptors, the antagonist potency profile for CIS-COS does not correspond exactly to any known A2 receptor subtype, suggesting that the cone receptor may be a novel A2 subtype. Our findings are consistent with previous reports that retinal adenosine levels are higher in the dark, and further suggest that adenosine could act as a neuromodulatory "dark signal" influencing photoreceptor metabolism and function in the fish retina.     

Combined effect of high-fat diet and copper deficiency during gestation on fetal copper status in the rat

We have previously shown that a low-copper (Cu) diet produced alterations in placental Cu transport and fetal Cu stores. Because Cu deficiency has been associated with lipid deposition in rat dam liver, we hypothesized that a high fat intake, a prevalent dietary habit in many populations, may worsen fetal Cu status and its closely linked iron (Fe) deposits. Pregnant rats were fed one of four diets during the second half of gestation: NFNCu: normal fat (7%), normal Cu (6 mg/kg); HFNCu: high fat (21%), normal Cu; NFLCu: normal fat, low Cu (0.6 mg/kg), and HFLCu: high fat, low Cu. One day before delivery, dams were anesthetized, and maternal as well as fetal plasma and tissues were obtained. Maternal, fetal, and placental weights were indistinguishable regardless of the group. Dam plasma Cu and placental Cu were lower in both LCu groups than in the NFNCu or the HFNCu groups. However, fetal plasma Cu was similar in all treatment groups. Dam and fetal liver Cu stores were reduced in the LCu groups compared to the NCu groups. This resulted in lower fetal/maternal liver Cu ratios in the NFLCu (1.79 ± 0.14,p < 0.05) and HFLCu (1.59 ± 0.21,p < 0.05) as compared to the NFNCu (4.12 ± 0.44) and the HFNCu (4.15 ± 0.27). Dam liver Fe was higher in the NFNCu than in HFNCu group (1.10 ± 0.8 vs. 0.89 ± 0.06 μmol/g,p < 0.05); fetal liver Fe from HFNCu and NFLCu dams was lower than that from NFNCu fetuses (NFNCu: 2.42 ± 0.14; HFNCu: 1.92 ± 0.15,p < 0.05; NFLCu: 1.81 ± 0.10,p < 0.01). Fetuses of the HFLCu group had a lower heart Fe than the NFNCu group (0.56 ± 0.03 vs. 44.0 ± 3.0 μg/g,p < 0.01). These data indicate that a maternal high-fat diet can potentially aggravate the effects of Cu deficiency by further altering fetal Cu and Fe tissue stores.     

Isolation and regulation of piglet cardiac AMP deaminase

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a K_m of 2 mM for 5-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the K_m of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD⁺, whereas, the K_m was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in V_max with no change in K_m. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.     

Identification and molecular cloning of a functional GDP-fucose transporter in Drosophila melanogaster

Nucleotide sugar transporters play a central role in the process of glycosylation. They are responsible for the translocation of nucleotide sugars from the cytosol, their site of synthesis, into the Golgi apparatus where the activated sugars serve as substrates for a variety of glycosyltransferases. We and others have recently identified and cloned the first GDP-fucose transporters of H. sapiens and C. elegans. Based on sequence similarity, we could identify a putative homolog in Drosophila melanogaster showing about 45% identity on protein level. The gene (CG9620) encodes a highly hydrophobic, multi-transmembrane spanning protein of 38.1 kDa that is localized in the Golgi apparatus. In order to test whether this protein serves as a GDP-fucose transporter, we performed complementation studies with fibroblasts from a patient with LADII (leukocyte adhesion deficiency II) which exhibit a strong reduction of fucosylation due to a point mutation in the human GDP-fucose transporter gene. We show that transient transfection of these cells with the Drosophila CG9620 cDNA corrects the GDP-fucose transport defect and reestablishes fucosylation. This study gives experimental proof that the product of the in silico identified Drosophila gene CG9620 serves as a functional GDP-fucose transporter.     

Theoretical and experimental analysis of analyte transport in a fiber-optic, protein C immuno-biosensor

Protein C (PC) is an important anticoagulant in human blood plasma, and early diagnosis of PC deficiency is critical for preventing dangerous thromboembolic complications. A fiber-optic PC immuno-biosensor has been under development in our research group for real-time PC-deficiency diagnosis. The sensor has demonstrated a good sensitivity and specificity for quantifying PC in buffered solutions. However, for plasma samples, with a limited sample reaction time, the sensor produced only 30% of the signal intensity of PC in buffer. The high plasma viscosity (1.9 cP) was speculated as the major reason for signal intensity reduction. In this investigation, the sensing performance of the fiber-optic PC biosensor is systematically characterized in terms of physical and chemical properties of the sample media. Theoretical and experimental analyses indicate that the reduced diffusion rate of PC molecules in viscous samples caused the sensing system to be more mass-transfer-limited. Convective flow of sample/reagent solutions during immunoreactions can increase the rate of the analyte mass transport from the bulk solution to the sensor surface, with reaction kinetics changing from mass-transfer-limited to reaction-limited as flow velocity increases. It was shown that PC sensor performance was significantly improved for plasma samples with convection. The effect of the flow velocity and incubation times for samples and reagents on the sensor performance was also systematically analyzed to optimize the assay protocol for PC sensing. Currently, a 6-cm-long immuno-biosensor is capable of quantifying PC in plasma (1 mL) in the heterozygous PC deficiency range (0.5 to 2.5 microg/mL) within 5 minutes, at an average signal-to-noise ratio of 50.     

Functional consequences of an in vivo mutation in exon 10 of the human GLUT1 gene

The functional consequences of an in vivo heterozygous insertion mutation in the human facilitated glucose transporter isoform 1 (GLUT1) gene were investigated. The resulting frameshift in exon 10 changed the primary structure of the C-terminus from 42 in native GLUT1 to 61 amino acid residues in the mutant. Kinetic studies on a patient's erythrocytes were substantiated by expressing the mutant cDNA in Xenopus laevis oocytes. K(m) and V(max) values were clearly decreased explaining pathogenicity. Targeting to the plasma membrane was comparable between mutant and wild-type GLUT1. Transport inhibition by cytochalasin B was more effective in the mutant than in the wild-type transporter. The substrate specificity of GLUT1 remained unchanged.     

Current knowledge of iron metabolism

Iron plays many roles in human physiology. In this article, we summarize the basic and current knowledge of this essential micronutrient on human metabolism.