External Label Research Articles

The fungal sheath of ectomycorrhizal pine roots: an apoplastic barrier for the entry of calcium, magnesium, and potassium into the root cortex?

The apoplastic permeability of the fungal sheath of two different ectomycorrhizal associations of Pinus sylvestris L. was analysed by laser microprobe mass analysis (LAMMA) and energy-dispersive X-ray spectroscopy (EDXS) after stable isotope labelling with 25Mg, 41K and 44Ca. Entry of 25Mg and 44Ca into the outer cortical apoplast of non-mycorrhizal roots was detected after 4 min of labelling. After a longer exposure time the endodermis with its Casparian band acted as an efficient apoplastic diffusion barrier for the radial movement of 25Mg and 44Ca into the stele. A fraction of approximately one-third of the apoplastic cations of the root cortex could not be exchanged against the external label even after longer exposure times. The ectomycorrhizal sheath of the two fungal species used, Pisolithus tinctorius (Pers.) Coker & Couch and Suillus bovinus (L. ex Fr.) Kuntze, does not completely inhibit the apoplastic movement of ions into the mycorrhizal root cortex, but retarded the penetration of isotopes into the cortical apoplast. In roots inoculated with S. bovinus, a clear labelling of the cortical apoplast could first be detected after 24 h of exposure to the stable isotope solution. At this time the labelling of the cortical apoplast in these mycorrhizal roots was higher than those of non-mycorrhizal roots and, with EDXS, changes in the element composition of the apoplast were detected. The results indicated that possibly hydrophobins localized in the fungal cell wall might be involved in the increased hydrophobicity of mycorrhizal roots and the lower permeability of the ectomycorrhizal sheath.

Thylakoid volume, proton translocation and buffering capacity as measured with spin-label techniques

In model studies with lipid vesicles it is shown that the main population of the spin label Tempamine is bound to the membrane surfaces, the piperidine ring directed to the lipid phase. The signal of external label but not of label in the surface is broadened by chromium oxalate. Inner volumes of vesicles can be derived from the partially resolved polar part of the high-field line of Tempone or from the area of the Tempamine spectrum removed by making chromium oxalate enter the vesicles. If this membrane-associated population is corrected for, rotational correlation times for the label in the lumen can be obtained showing that hindrance of rotation is only by a factor of 3–5 instead of 10 as previously reported. In studies with thylakoids volumes of 1–3 μl/mg Chl were found with 0.3 M sorbitol as an osmoticum, 5 mM MgCl 2, 20 mM KCI, and 20 mM chromium oxalate. The internal buffering capacity and the magnitude of pH changes in the inner volume can be determined from flash-induced changes in the amine distribution. The buffering capacity is found to be 7–20 mM in buffer-permeable and approx. 100 mM in buffer-impermeable thylakoids, that is approx. 100 neq per mg Chl. The apparent H +/e −-value in impermeable preparations was found to be up to 0.7 and lower in permeabilized material. ΔpH per flash is 0.04–0.06 units. Possible sources of errors, particularly the presence of non-functional or non-thylakoid membranes, are discussed. Time-resolved signals are presented and several side effects and their suppression are discussed. The response time of the method is up to 2 ms, protons from the donor side of Photosystem II can be separated kinetically from those liberated by the intersystem chain. While transients with less than 2 ms and approx. 20 ms were found with ferricyanide as an electron acceptor in accordance with the results with neutral red, pronounced slow phases ( t 1 2 is several hundred ms) were found without acceptors. Evidence is presented indicating that at least part of these responses do not originate from the thylakoid inner volume.

Age-dependent decrease of the lateral diffusion constant of proteins in the plasma membrane of hepatocytes as revealed by fluorescence recovery after photobleaching in tissue smears

When fresh liver is smeared on slides and incubated in Krebs-Henseleit Ringer solution containing 1 mM H 2O 2 at 37°C, a yellowish-green autofluorescence develops in the hepatocyte plasma membrane. Indirect evidence shows that this peroxide-induced autofluorescence (PIAF) is due most probably to chemical reactions between proteins and the malondialdehyde produced by the membrane lipid peroxidation. Although the chemical nature of the PIAF has not been clarified yet, it is suitable under certain conditions for the measurement of the average lateral diffusion constant of the membrane proteins by means of the fluorescence recovery after photobleaching (FRAP) technique without the addition of any external fluorescent label. Analysis of four age groups for both sexes of Fischer 344 rats (3–5 rats per group, total 32) from 2 to 31 months of age revealed a significant negative linear correlation of the lateral diffusion constant of proteins with age in both sexes, with the slope of the females being somewhat smaller. Young males showed a diffusion constant about 2.8 × 10 −10 decreasing to 1.7 × 10 −10 cm 2·s −1 by 31 months of age at 37°C, whereas the respective values in females were 2.7 × 10 −10 and 1.9×10 −10. The results are consistent with the predictions of the membrane hypothesis of aging, according to which an age-dependent loss of the passive permeability of the cell membrane for potassium (and probably for water) is the crucial point of the cellular aging.

A new spin label method for the measurement of erythrocyte internal microviscosity

A new spin-label method for the measurement of the internal microviscosity of erythrocyte is presented. The spin label used is 2,2′,5,5′-tetramethyl-3-maleimidopyrrolidinyl- N-oxyl (MAL-5) which penetrates inside the red blood cell and binds covalently on cytoplasmic glutathione. After washing off the external label, 98% of the electron paramagnetic signal is due to the labelled glutathione. This signal allows one to measure the rotational correlation time of the label. A calibration curve established with spin-labelled glutathione in sucrose solutions of increasing viscosity is used to convert the measured rotation times into viscosity units. This method avoids the use of unphysiological salts like potassium ferricyanide, and permits the study of red blood cells in various suspension media. In normal human subjects, the mean value of microviscosity is 4.45 ± 0.16 mPa·s at 20°C in isotonic saline (25 subjects) and 6 ± 0.25 mPa·s in plasma. The variations of microviscosity as a function of the osmolarity of the medium are explained according to a theoretical model taking into account the variations of the red blood cell volume and the viscometric properties of haemoglobin.

Protein synthesis in pulmonary alveolar macrophages Source of amino acids for leucyl-tRNA

Extracellular, intracellular and tRNA-bound leucine pools of the adherent pulmonary alveolar macrophage were examined to determine the relationships between them and the precursor for protein synthesis. When cells were cultured in media of various leucine concentrations, the patterns of isotope distribution in intraceullar and extracellular leucine did not correlate with the patterns seen in protein-bound leucine. Hence, the free leucine pools cannot be used reliably as precursors for calculating rates of protein synthesis. tRNA-bound leucine, however, behaved isotopically as if it were the precursor. Constant synthetic rates were calculated using the tRNA specific activity over a wide range of leucine concentrations. In addition, by measuring the tRNA-bound specific activities of three different amino acids, leucine, valine and phenylalanine, and their respective specific activities in protein, we were able to calculate independently three separate but identical synthetic rates. At physiological amino acid concentrations, the macrophage intracellular leucine pool and the tRNA-bound leucine pool received less than half of their amino acids from extracellular sources. At 5 mM external leucine, the intracellular specific activity was indistringuishable from that of the medium leucine, but the specific activity of the tRNA-bound leucine pool remained only about 50% that of the extracellular value. The most straightforward interpretation of why the tRNA-bound leucine did not flood with external label under conditions where the intracellular pool has reached equilibrium is to propose that some portion of the leucine for protein synthesis is derived directly from protein turnover before the degradation products have mixed with the common amino acid pool.

Labeling and Isolation of Plasma Membranes from Corn Leaf Protoplasts

A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confirmed with the aid of an external label, diazotized [(125)I]iodosulfanilic acid. Gentle cell disruption enabled internal organelles to be maintained intact and thus facilitated separation from the plasma membrane. The plasma membrane-enriched fraction was devoid of chloroplast or mitochondrial markers, whereas markers for the endoplasmic reticulum and golgi indicated minimal contamination. The highly enriched plasma membrane fraction contained a Mg(2+)-dependent, K(+)-stimulated ATPase with a pH optimum near neutrality. The position of the membranes on sucrose density gradients indicates that the plasma membranes have characteristics similar to other plasma membrane fractions.

Studies on the uptake of xanthine by Schizosaccharomyces pombe from the stationary growth phase

1. Cells of Schizosaccharomyces pombe from the stationary growth phase show only a low capacity for the uptake of xanthine. In the presence of 2 muM xanthine, the initial uptake rate is found in the range of 6-10- minus 21 Moles times Cells- minus 1 times Min- minus 1 at 30 degrees C. 2. Preincubation in glucose solution results in a pronounced stimulation of the uptake rate. 3. The stimulating effect of the glucose pretreatment is repressed by cycloheximide, ammonium ions and amino acids. 4. The substrate dependency of the uptake rate shows a saturation kinetics. The Km-value has been determined to 6 muM. The temperature optimum is found in the range of 30 degrees C. 5. Xanthine is only slightly accumulated within the cells against the external concentration. Most of the external xanthine label is stored up in form of xanthosine and guanosine as well as incorporated in nucleic acids.

Changes in a surface-labelled galactoprotein and in glycolipid concentrations in cells transformed by a temperature-sensitive polyoma virus mutant.

IN hamster cells transformed by a temperature-sensitive mutant of polyoma virus we have found that the external label in a galactoprotein disappears and the cellular concentration of lactosylceramide increases when the cells are grown at the permissive temperature. These changes can be reversed at the non-permissive temperature.

Evidence for the Transfer of Rhizobial Metabolites to the Polyploid Nuclei of Young Clover Nodules

AbstractAutoradiography was used to provide evidence for the transfer of Rhizobium produced moieties to the host nuclei of young clover nodule cells. Cells of Rhizobium trifolii Dangeard were labeled with 3H‐adenine or 4,5‐3H‐l‐leucine and 3,4‐3H‐l.‐proline, washed free of external label, and allowed to nodulate young seedlings of white clover (Trifolium repens L. cv. White Dutch). Sections (0.5–1.0 μm) of young nodules up to four days old were autoradiographed using the dipping technique. Grain counts indicated movement of tritium from the leucine‐proline labeled rhizobia to the polyploid nuclei of two day old clover nodule cells. Acetylene reduction was not detected until approximately 24 hours after the transfer of tritium was observed. No transfer of tritium was observed with 3H‐adenine labeled rhizobia. It is hypothesized that nodulating rhizobia may induce clover nodule cells to initiate leghemoglobin synthesis by the transfer of a bacterially produced inducer.

View

The eternal argument about the overall pattern in which we organize our schools tends to induce a state of total complacency about what goes on inside them. The politicians prescribe the external label, but in Britain they have (very properly) no power to lay down much else. The curriculum, the ambience, the aims of the school are in the hands of the headmaster, and no one talks about them very much. Since to most parents the label is all, it is reputation rather than reality which matters. But in fact labels tell you little about the contents of the bottle. Antiquated educational ideas often flourish in supposedly revolutionized comprehensive schools, and some grammar schools have changed out of all recognition. The sooner the argument can be swung round to the purpose of education within the schools, the better.

Synthetic Polypeptide Metabolism

Summary The metabolic fate in rabbits of intravenously administered, iodine-labeled poly d-Glu55d-Lys39-d-Tyr6 differs markedly from that of its enantiomorph, poly Glu58Lys36Tyr6 (No. 2). Although both polymers are rapidly cleared from the circulation, 131I is slowly excreted in the urine after d-polymer injection, but quite rapidly excreted after l-polymer injection. Only 32% of the d-polymer is degraded by the 19th day, whereas 83% of the l-polymer is degraded by the 7th day. This delay in the degradation of the d-polymer is reflected in the retention of large amounts of the injected material in the liver and in the kidneys. The amount of d-polymer in the liver slowly declines from a maximum of 16% at 5 days, but the amount in the kidney continues to increase for 2 to 3 weeks and ultimately reaches 32% of the injected polymer. Radioautographic studies show that both polymers enter the proximal tubular cells of the kidney: the d-polymer persists, whereas the l-polymer is quickly eliminated. Studies with ureteral ligation to stop glomerular filtration demonstrate that the polymers are filtered through the glomerulus and then reabsorbed. Anephric animals show a slower serum elimination rate for the d-polymer than normal animals. Since the hepatic uptake is unchanged, it is likely that the kidney rapidly extracts a certain population of the d-polymer molecules which are not as readily taken up by the liver. Since ultracentrifugation of the labeled d-polymer prior to injection does not affect its distribution, the basis for this biologic heterogeneity is not aggregation. Chromatographic analyses of the dialyzable radioactivity in the urine and in the homogenates of the liver and kidney clearly show the presence of labeled peptides derived from the degradation of the d-polymer or of the l-polymer. A second external label, 59Fe-ferrocene, confirms the serum elimination and urinary excretion patterns obtained with iodine, and, in general, also confirms the organ retention pattern of the d-polymer. However, significant incorporation of the iron from the ferrocene into hemoglobin and other compounds occurs, and this fact limits the usefulness of the ferrocene label, especially for studies of tissue localization. The prolonged retention of the d-polymer in the organs and its gradual release over a long period of time are postulated to cause immunologic paralysis. This hypothesis explains the apparent lack of immunogenicity of d-polymers under the usual conditions of immunization and the lack of a significant booster effect and the ultimate disappearance of circulating antibody following repeated injections of small amounts of the d-polymer.

Studies in Vitro of the Transport of the A and B Chains of Insulin in Serum

(Received for publication, January 27, 1964) The introduction of an external radioisotopic label on the insulin molecule has permitted extensive invest.igations into the disposal of this polypeptide hormone in biological fluids (l-3). It has been observed that, following its injection into mammals, ‘311-insulin is rapidly modified as reflected by increased solubility of serum radioactivity in trichloroacetic acid and by the demon- stration of radioactivity migrating in the c,2-globulin and albumin regions on paper electrophoresis (4-6). It has also been shown that as a result of protracted incubation of 1311-insulin with serum at 37” in vitro, radiolabeled compounds occur with electrophoretic mobilities similar to those found in serum after administration of i3iI-insulin (5, 7). The raljid inactivation of insulin in vivo has been considered to result from disruption of this molecule by an intracellular enzyme with a major specificity for insulin (3, 8-11). Mirsky