The influence of spermine on the structural dynamics of yeast tRNAPhe

A tRNA^Phe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T₁–T₃) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T₁ and T₂, and a slow one (100–1000 ms) between T₂ and T₃. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T₃, as does Mg²⁺, but the final ratio $\text{[}\text{T}{}_2\text{]}\text{[}\text{T}{}_1\text{]}$ obtained with spermine is higher than with Mg²⁺, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

T-antigen expression by polyoma mutants with modified RNA splicing

Polyoma virus mutants were constructed that could not express all the three T-antigens. The mutagenesis was directed to the two 5' splice sites utilized in the maturation of early RNA. The mutant bc1051 had a base change at the splice site of large T-antigen mRNA, and the mutants dl1061 and dl1062 had deletions at the corresponding splice point of small and middle T-antigen mRNA. The site was removed in mutant dl1061 and altered by fusion to upstream sequences in mutant dl1062. Analysis of viral RNA showed that dl1061 and dl1062 formed only large T-antigen mRNA, whereas bc1051 did not produce this RNA-species. However, the biological properties of dl1062 suggested that it also produced mRNA directing the synthesis of a small T-antigen-related polypeptide, at least in low amounts. Only mutant bc1051 could induce transformation of rat cells. In mouse 3T3 cells dl1062 multiplied to a limited extent, while bc1051 and dl1061 failed to produce virus. However, dl1061 DNA was synthesized at a low rate which could be increased to normal levels by co-transfection with mutant bc1051. This result suggests that polyoma small and middle T-antigen have a previously unrecognized function in the early phase of the infection process, or in viral DNA-synthesis.     

Immunoglobulin synthesis and glucose-6-phosphate dehydrogenase as cell markers in human lymphoblastoid cell lines

Multiple lymphoblastoid cell lines were established from each of seven Burkitt lymphoma biopsies and from tonsils, removed from four patients with chronic tonsillitis. The cellular origin of the lines was studied using as markers the pattern of immunoglobulins secreted into the medium and the cells' glucose-6-phosphate dehydrogenase (G-6-PD) phenotypes.Lines from the same tonsil biopsy differed from each other by their patterns of immunoglobulin synthesis and G-6-PD phenotypes. All tonsil-derived lines secreted complete immunoglobulins. Newly established lines usually produced several heavy and light chain types, indicating multicellular origin, but the number of components produced decreased during the course of long-term cultivation. G-6-PD phenotypes of lines established from the same tonsil removed from a G-6-PD heterozygote differed—B, A and B/A phenotypes were found. The B/A lines rapidly changed to a single enzyme phenotype (B or A) when maintained in culture.The immunoglobulin and G-6-PD phenotypes in lines derived from Burkitt lymphomas differed from those of tonsil lines in several respects: (1) Some lines produced no immunoglobulins; (2) in immunoglobulin-synthesizing lines, the patterns of heavy and light chain production were more restricted than in tonsil lines; (3) after some months in culture, a uniform pattern of immunoglobulin synthesis was found in all lines derived from the same tumour; (4) lines from G-6-PD heterozygotes had the same single enzyme phenotypes as were found in the tumours.The data strongly suggest that most lines from Burkitt lymphomas are derived from the tumour clones and that most tonsil-derived lines have multicellular origin.     

Observations on the cellular localization of dopamine in the caudate nucleus of the rat

Summary The cellular localization of dopamine in the caudate nucleus of the rat hat been studied with the highly sensitive and specific fluorescence method of Falck and Hillarp, and by electron microscopy. The histochemical studies provided strong support for the view that the dopamine is concentrated within very fine nerve fibres which have abundant varicosities with an intense fluorescence. The electron microscopical studies revealed the presence of a tightly packed plexus built up i.a. of abundant synaptic nerve terminals, many of which had a diameter below 0.4 . The terminals made synaptic contact mainly with processes that seemed to belong to an extensive dendrite net.The investigation was supported by research grants from the United States Public Health Service (02854-04), The Swedish Medical Research Council and the Knut and Alice Wallenberg Foundation.     

Reversed-phase liquid chromatographic determination of idarubicin and its 13-hydroxy metabolite in human plasma

A method is given for the determination of idarubicin and its main metabolite, idarubicinol, in plasma from cancer patients. Idarubicin and idarubicinol are extracted from 2-ml samples of buffered plasma (pH 8.1) using chloroform-1-heptanol (9:1). After reextraction into phosphoric acid (0.1 M), separation is performed by reversed-phase liquid chromatography on a LiChrosorb RP-2 column (5 μm) with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The absolute recovery in the range 5–100 ng/ml was greater than 83% with a precision better than 8% (relative standard deviation), using photometric detection at 484 nm. Proper handling of whole blood samples containing idarubidin is essential to avoid metabolic conversion into idarubicinol. Prolonged storage of the drug and its main metabolite under alkaline conditions should be avoided to prevent chemical degradation.     

Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH-regulated channel mediating iodide efflux selectively across the apical domain of the plasma membrane.

The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid-stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle.     

Conversion of fat into yeast biomass in protein-containing waste-water

Summary Candida tropicalis S001 was grown on the lipid fraction of a protein-containing waste-water in order to (i) remove fat from the water, and (ii) produre yeast biomass for feed. The yeast cells were separated from the waste-water by sedimentation. Defatted waste-water was used for methane production and gave a yield of a 0.3 m³ methane/kg reduced chemical oxygen demand. The maximum specific growth rate (µ_max) of C. tropicalis growing on waste-water fat at pH 4.0 was 0.35 h^–1; the fat content was decreased from 8 g/l to about 0.1 g/l within 24 h. In continous culture a corresponding reduction was maintained at dilution rates up to 0.36 h^–1. The effect on growth of pH, temperature and CO₂ concentration was studied with triolein as the major carbon source. The µ_max was nearly constant (0.16 h^–1) in the pH and temperature range of 3.2–4.0 and 30°–38° C, respectively; 10% CO₂ was optimal for growth. Growth on triolein resulted in a biomass yield of 0.70 g dry weight/g fat. Offprint requests to: S. Rydin     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.