Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

Gene expression during induction of somatic embryogenesis in carrot cell suspensions

We report the identification, via their cDNAs, of genes which are temporarily transcribed during the initiation of somatic embryogenesis in carrot (Daucus carota L.) cells cultured in an auxin-free medium. Their expression is roughly associated with the first morphogenetic, or globular, stage. A cDNA library ( gt 10) was established using poly(A)⁺ -rich RNAs from cells deprived of auxin for 8 d. By differential screening a number of clones corresponding to early-induced embryogenic genes were identified. For several a temporary accumulation of the specific mRNA between 6 and 16 d after induction was observed. With regard to the nucleotide sequence and the respective deduced amino-acid sequence, two glycine-rich proteins and a polypeptide with a proline-rich domain were among the products of genes activated at the onset of somatic embryogenesis.Abbreviations b, bp bases, basepairs - 2,4-D 2,4-dichlorophenoxyacetic acid Sequence data reported here will appear in the EMBL Genbank and DDBJ Nucleotide Sequence Databases under the following accession numbers: X 15436 for clone DC 2.15 (proline-rich protein), X 15706 for clone DC 7.1 (glycine-rich protein, DCGRP) and X 14067 for clone DC 9.1 (glycine-rich protein, DCGRP)This research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance.     

Seleno-DL-methionine and the protection of human platelets against freezing injury

The effect of seleno-DL-methionine, which has antioxidative properties, on the recovery of human platelets after freezing with 0.5 mol/liter glycerol or 0.7 mol/liter (5% v/v) dimethyl sulfoxide was investigated. Incubation of platelets with 2 mumol/liter seleno-DL-methionine for 30 min at room temperature before equilibration with the protective additives improved the post-thaw uptake of 5-hydroxytryptamine and the percentage reversal in the hypotonic stress test. The effect was small, but in view of the ability of seleno-DL-methionine to inhibit lipid peroxidation in membranes, the results suggest that oxidative damage is implicated in freezing injury. The dimethyl sulfoxide protocol apparently afforded greater protection to the platelets than the glycerol protocol. But, if the platelets were incubated for 24 hr at 37 degrees C after thawing, there was a marked improvement in the response of cells in the hypotonic stress test, particularly in the samples frozen with glycerol, and there was no longer any difference between the two additives. There was, however, a concomitant loss of almost half the number of cells in the thawed suspensions during the prolonged incubation at 37 degrees C.     

Inhibition of sugar uptake by ascorbic acid in Escherichia coli

The uptake of glucose by the glucose phosphotransferase system in Escherichia coli was inhibited greater than 90% by ascorbate. The uptake of the nonmetabolizable analog of glucose, methyl-alpha-glucoside, was also inhibited to the same extent, confirming that it was the transport process that was sensitive to ascorbate. Similarly, it was the transport function of mannose phosphotransferase for which mannose and nonmetabolizable 2-deoxyglucose were substrates that was partially inhibited by ascorbate. Other phosphotransferase systems, including those for the uptake of sorbitol, fructose and N-acetylglucosamine, but not mannitol, were also inhibited to varying degrees by ascorbate. The inhibitory effect on the phosphotransferase systems was reversible, required the active oxidation of ascorbate, was sensitive to the presence of free-radical scavengers, and was insensitive to uncouplers. Because ascorbate was not taken up by E. coli, it was concluded that the active inhibitory species was the ascorbate free radical and that it was interacting reversibly with a membrane component, possibly the different enzyme IIB components of the phosphotransferase systems. Ascorbate also inhibited other transport systems causing a slight reduction in the passive diffusion of glycerol, a 50% inhibition of the shock-sensitive uptake of maltose, and a complete inhibition of the proton-symport uptake of lactose. Radical scavengers had little or no effect on the inhibition of these systems.     

Surface-charge related actions of polylysine on thylakoid membranes

Polycation binding to the negatively charged surface of chloroplast thylakoid membranes is known to cause an inhibition of photosystem I activity. It also interferes with the cation-dependent rearrangement of chlorophyll proteins in the thylakoid membrane. It was shown that added anions prevented or reversed the inhibition of photosystem I by polylysine without decreasing its binding to the membranes. Anions also caused a change in the interaction of the chlorophyll proteins in polylysine-treated thylakoids as indicated by an increase in the relative fluorescence intensity from photosystem II. In both cases, the relative effectiveness of the anions tested depended on their valence; for example, the tetravalent species Fe(CN)₆^4t- was effective at a concentration at least 2 orders of magnitude lower than the divalent species SO₄^2?. These results suggest that anions act by screening the positive charge of the polylysine-coated membrane surface. Measurements of the response of the anionic fluorescent probe 1-anilinonapthalene-8-sulfonate to an addition of anions to polylysine-treated thylakoids supported this contention. It was concluded that the action of polylysine on photosystem I and on the chlorophyll proteins is mediated by changes of the electrical properties of the thylakoid membrane and may not involve a direct binding of the polycation to the affected membrane proteins.     

Determination of Root Exudates in a Steril Continuous Flow Culture. II. Short-Term and Long-Term Variations of Exudation Intensity

The exudate production of alfalfa under the conditions of the sterile flow culture was quantitatively measured. In the first 40 days 3.10⁻³ μmoles amino-N, 2.5 μequivalents of organic acids and approximately 10⁻⁴ μmoles of reducing sugars were liberated per plant and per day into the percolating nutrient solution. The amino acid concentration in the outflow varies according to a daily periodicity. The exudation of a colored substance also shows daily periodical variations. This pattern is different from the pattern of the amino acid exudation, however, and directly coupled to shoot illumination. Short-term 2,4-dinitrophenol additions to the nutrient lower the liberation of amino acids into the percolating solution.     

The enzymic estimation of glutamate and glutamine

A method of estimating glutamic acid is described, based on its dehydrogenation by glutamate dehydrogenase coupled, by means of N-methylphenazine methosulphate, to the reduction of tetrazolium salts. The method is suitable for the estimation of 0-0.3mumole of glutamic acid. The response is linear, but not stoicheiometric: possible reasons for this are discussed. If suitable precautions are taken, the use of a partially purified preparation of glutaminase makes it possible to estimate glutamine also.     

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