Superoxide perturbation of the organization of vascular endothelial cell membranes

Cultured porcine thoracic aorta endothelial cells were covalently labeled with 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Electron paramagnetic resonance spectrometry revealed two major binding environments representing strongly and weakly immobilized species. The disorder parameter of weak/strong, determined from the respective peak amplitudes, was irreversibly elevated following incubation of endothelial cells with a superoxide-generating system, indicating increased membrane fluidity. The rate of increase in membrane disorder was dependent upon superoxide generation rates. Incorporation of the spin-label at concentrations less than 250 microM had no effect on cell viability. The cellular proteins reacting with the spin-label were predominantly membrane proteins, characterized by immunoblotting using a rabbit anti-4-maleimido-2,2,6,6-tetramethylpiperidinooxyl IgG, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophorectic transfer to nitrocellulose.     

Sequence Determinants of GLUT1 Oligomerization: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS*

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.     

Effect of the ionic environment and of various neurotransmitters on spontaneous motility in snail intestine (author's transl)]

A spontaneous rhythmic motility of the isolated intestine in snail, Chryptomphalus hortensis, has been registered at 30 degrees C with the aid of an electronic transductor and an appropriate amplification. These slow regular movements are affected by ionic composition changes in the suspension medium. Na+, K+ and Ca++ ions prove to be important in this motility, and the addition of Ba++ markedly stimulates it. ACh produces hypermotility from 1.8 X 10(-11) g/ml. Its effect decreases in the presence of atropine and increases in that of pyridostigmine. The intestine is sensitive to 5-HT from 10(-10) g/ml, which stimulates its activity. The effect of histamine is weak. Low concentrations of adrenaline tend to increase the amplitude, whereas concentrations from 10(-5) g/ml onward produce a cease of motility in relaxation.     

Nucleotide sequence of the second psbG gene in Synechocystis 6803. Possible implications for psbG function as a NAD(P)H dehydrogenase subunit gene

Nucleotide sequencing of the second Synechocystis 6803 psbG gene, psbG2 shows the predicted polypeptide to be 219 amino acids long. It is less similar to chloroplast psbG genes than is the Synechocystis psbG1 copy. Alignment with seven other psbG protein sequences, including that from the Paramecium mitochondrial genome reveals a central highly conserved region common to each. This is discussed as evidence supporting the proposal that the psbG polypeptide is a NAD(P)H dehydrogenase (complex I) subunit in cyanobacteria, chloroplasts and mitochondria.     

Spiggin levels are reduced in male sticklebacks infected with Schistocephalus solidus

Using an enzyme-linked immunosorbent assay (ELISA) based technique, Schistocephalus solidus infection was shown to considerably reduce levels of spiggin in the kidney of male three-spined sticklebacks Gasterosteus aculeatus from an oligotrophic upland lake. These results suggested a graduated effect of the infection on the reproductive physiology of male three-spined sticklebacks.