Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.     

Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.     

Role of prostaglandin endoperoxides in the serum thiobarbituric acid reaction

In the presence of heme and reduced glutathione, prostaglandin (PG) endoperoxides underwent rapid conversion to malondialdehyde and 12l-hydroxy-5,8,10-heptadecatrienoic acid. In addition, PG endoperoxides as well as lipid peroxides produced malondialdehyde to yield a red pigment during the thiobarbituric acid reaction with different efficiencies. The relative rates of the reaction were: 1,1,3,3-tetraethoxypropane, 100; PGG₂, 55; PGH₂, 32; and 15-hydroperoxyarachidonic acid, 6. The thiobarbituric acid reactive materials in rabbit serum decreased by 25–60%, after intravenous administration of aspirin (a cyclo-oxygenase inhibitor) and with a concomitant decline of serum PG levels. These results, taken together, suggested that serum thiobarbituric acid values, considered to be an indicator of lipid peroxide levels, were to a significant extent due to PG endoperoxides and their derivatives.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.     

Stimulating factor for calf thymus DNA polymerase β

A factor that stimulates purified DNA polymerase β about 2-fold was separated from DNA polymerase β activity on a DNA-cellulose column. During the early stage of purification, the factor may be associated with DNA polymerase β to form a complex that sediments at 3.9 S in sucrose gradients and behaved as a 52,000 dalton protein on a Sephadex G-100 column. The complex, which contains 40,000 and 12,500 dalton polypeptides, was insensible to the stimulator, and did not show any exonuclease activity.     

A contractile ring and cortical changes found in the dividing Tetrahymena pyriformis

A contractile ring consisting mainly of microfilaments was found in the fission zone of dividing Tetrahymena pyriformis. Diameters of the microfilaments were widely distributed from 2.5 to 15 nm. Ring-associated structures such as lateral stripes, linkers and beads with siender tails were recognized. Lateral stripes arranged at regular intervals of about 84 nm on some parts of microfilament bundles were found in both tangential and transverse sections, suggesting that they correspond to bands fastening the contractile ring microfilaments. Linkers that connect individual lateral stripes to the epiplasmic layer were present. Beads or beads with slender tails were found to be arranged on some microfilaments.The results of the present paper also indicate that drastic morphological changes occur in the cortex of the fission zone, especially in the epiplasmic layer, accompanying contraction of the division furrow. The epiplasmic layer which was proved to be a compact filamentous network in this study has been known to exist at the periphery of cytoplasm in immediate contact with one of the cell surface membranes, the inner alveolar membrane; however, in the fission zone of the dividing ceil, it was frequently separated from the membrane and subsided into the cytoplasm. The subsided epiplasmic layer was then loosened and dispersed. The subsidence of the epipiasmic layer appears to be caused by the force generated by the contraction of the contractile ring and transmitted with the linkers to the epiplasmic layer. The changes observed in the epiplasmic layer are presumably indispensable for the rigid cortical layer contraction involved in cytokinesis of Tetrahymena.     

An improved method based on the Porter-Silber reaction for determining 17-hydroxycorticosteroids in urine

This paper describes a highly specific method for determining urinary 17-hydroxycorticosteroids, which has been developed by (i) changing the composition of the Porter-Silber reagent and (ii) removing contaminants interfering with the color reaction by addition of sodium bisulfite to β-glucuronidase-hydrolyzed urine before extraction with solvent. For a reference method the Norymberski-Riondel (J. K. Norymberski and A. Riondel 1970, Biochem. J. 120, 493–498) gas chromatography (glc) was used: Correlation coefficient between the present method and GLC = 0.988, deviation from the theoretical regression LINE = 6.8%, and coefficient of SIMILARITY = 0.56. These results are much better than those obtained by I. Ernest, B. Håkansson, J. Lehmann, and B. Sjögren (1964, Acta Endocrinol. 46, 552–562) for the original Porter-Silber method in comparison with the chromatographic measurement of grouped and individual steroids.