Mechanism of effect of aging on membrane transport in leaf strips of Centranthus ruber: Possible ethylene involvement in cutting shock

Cerulenin has been used to investigate the mechanism by which leaf strips of Centranthus ruber (L.) Lam et D.C. increase their capacity for solute uptake during a period of incubation in CaSO₄ (aging). -Aminoisobutyric acid was used to assess uptake capability. The leaf strips developed their uptake capacity for at least 8–10 h after excision. Cerulenin, if added to the aging medium immediately after cutting or at any time during the aging process, almost completely halted this development, but did not bring about loss of the uptake capacity already achieved. That cerulenin was specifically interfering with fatty-acid biosynthesis was indicated by the fact that it drastically depressed incorporation of labelled acetate into the lipid fraction but did not affect the incorporation of labelled alanine. Cycloheximide strongly inhibited the development of uptake capacity, but sensitivity to actinomycin D was not evident for at least 2 h. The results are consistent with the concept that slicing leads to immediate damage to tissue membranes and that aging is a process of membrane repair, involving renewed synthesis of membrane lipids and membrane proteins.The damage to membranes associated with cutting may involve wound ethylene. This is indicated by the following findings: Treatment of aged leaf strips with Ethrel (2-chloroethylphosphonic acid) resulted in drastic loss of their acquired uptake capacity. The strips recovered from such Ethrel-induced loss of uptake capacity while aging in CaSO₄ as they can after cutting. Cerulenin halted recovery of uptake capacity after ethrel treatment just as it did after cutting. Treatment of leaves before cutting with amino-ethoxyvinylglycine somewhat improved the uptake performance of leaf strips immediately after excision.     

Isolation and identification of the principal siderophore of the plant pathogenic fungusBotrytis cinerea

Summary The plant pathogenic hyphomyceteBotrytis cinerea has been shown to produce several trihydroxamate siderophores under conditions of low-iron stress. The total siderophores amounted to approximately 30 mg/l culture filtrate after 5 days of incubation in an asparagine/salt/glucose medium. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) on a reversed phase indicated that ferrirhodin is the predominant siderophore of this fungus. Chemical characterization of the principal siderophore by fast-atom-bombardment (FAB) mass spectrometry, nuclear magnetic resonance (¹H-NMR,¹³C-NMR) and comparison with a reference revealed the identity with ferrirhodin. NMR studies performed on desferrirhodin (desferrirhodin) in dimethylsulfoxide and water revealed the existence of two conformers in D₂O resulting from acis-trans isomerization of the hydroxamic acid groups. Comparative iron-uptake studies showed the following order of uptake inB. cinerea: ferrichrysin (100%), ferrirubin (57%), ferrirhodin (45%), hexahydroferrirhodin (45%), coprogen 6%. Concentration-dependent uptake of ferrirhodin resulted in saturation kinetics only in the low concentration range of 0–30 M (K _m = 2.5 M,V _max = 80 pmol min^–1 mg(^–1). A non-saturable, linear uptake was observed in the high concentration range of 30–80 M. The low concentration range appears to be the physiologically significant range, where siderophore-mediated iron transport inB. cinerea occurs.     

Synthesis and Structural Analysis of Oxadiazole Carboxamide Deoxyribonucleoside Analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2′-deoxy-3′,5′-β-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2′-endo (“south”) conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.

     

Calculating spatial statistics for velocity jump processes with experimentally observed reorientation parameters

Mathematical modelling of the directed movement of animals, microorganisms and cells is of great relevance in the fields of biology and medicine. Simple diffusive models of movement assume a random walk in the position, while more realistic models include the direction of movement by assuming a random walk in the velocity. These velocity jump processes, although more realistic, are much harder to analyse and an equation that describes the underlying spatial distribution only exists in one dimension. In this communication we set up a realistic reorientation model in two dimensions, where the mean turning angle is dependent on the previous direction of movement and bias is implicitly introduced in the probability distribution for the direction of movement. This model, and the associated reorientation parameters, is based on data from experiments on swimming microorganisms. Assuming a transport equation to describe the motion of a population of random walkers using a velocity jump process, together with this realistic reorientation model, we use a moment closure method to derive and solve a system of equations for the spatial statistics. These asymptotic equations are a very good match to simulated random walks for realistic parameter values.     

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum

Summary. Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria. Correspondence and reprints: Department of Cytology and Histology, Faculty of Biology and Soil Sciences, Saint Petersburg State University, Universitetskaya naberezhnaya 7/9, 199034 Saint Petersburg, Russia.     

Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDP<-->ADP+(d)NTP. This reaction, suggested to occur by the transfer of the gamma-phosphoryl from ATP to the nucleoside diphosphate, is overall similar to that normally carried out by nucleoside diphosphate kinase (Ndk). Accordingly, Adk was proposed to be responsible for residual Ndk-like activity measured in a mutant strain of Escherichia coli, where the ndk gene was disrupted. We present data supporting a mechanism for the synthesis of nucleoside triphosphates by Adk that unlike the previously suggested mechanism mentioned above are in complete agreement with the current knowledge about the Adk enzyme and its various catalytic properties. We propose that nucleoside triphosphate synthesis occurs by beta-phosphoryl transfer from ADP to any bound nucleoside diphosphate. Our results point to the fact that the proposed Ndk-like mechanism of Adk originated from an erroneous interpretation of data, in that contamination of ATP preparations with AMP and ADP was not taken into account. Our results also address the proposed role of Adk in restoring a normal growth rate of mutant strains of E. coli lacking Ndk. These mutant strains apparently, in spite of a mutator phenotype, are able to synthesise nucleoside triphosphates by alternative pathways to maintain the same growth rate as the wildtype.     

The Effect of the Gamma Modulator on Na/K Pump Activity of Intact <Emphasis Type="Italic">Mammalian Cells</Emphasis>

This study concerns the modulatory effects of the gamma modulator of the Na/K pump, in particular whether the effects seen in previous experiments with isolated membranes are relevant to Na/K pump behavior in intact mammalian cells. For this purpose, HeLa cells previously transfected with the rat Na/K catalytic subunit were used. The results show that both variants of the regulator, γa and γb, decrease the apparent affinity of the pump for Na⁺ and cause a modest increase in apparent ATP affinity as seen in measurements of ouabain-sensitive ⁸⁶Rb(K⁺) influx into cells in which ATP was varied using antimycin A and glucose. Equivalent results had been obtained previously in our analyses of Na,K-ATPase activity of membrane fragments, i.e., an increase in K_0.5(Na) at high K⁺ concentration and a decrease in K′_ATP. Comparison of clones of γ-transfected and mock-transfected cells (with similar V_max values) indicated that γ causes a modest ≈30% increase in the steady-state concentration of intracellular Na⁺. Furthermore, for both γa and γb, values of intracellular Na⁺ were similar to those predicted from the kinetic constants, K_0.5(Na) and V_max. Finally, there was a γ-mediated increase in apparent affinity for extracellular K⁺, which had not been detected in assays of permeabilized membranes.