Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.1-180 microM, of oxygen concentrations. These cells lost the ability to produce cyanide upon aerobic incubation in the absence of both the carbon energy source (L-glutamate) and the metabolic precursor of hydrogen cyanide (glycine). This loss of cyanogenesis was dependent on oxygen concentration; 1.0 microM oxygen produced no detectable loss, whereas 180 microM oxygen caused a rapid decline in cyanogenic ability. The endogenous cyanide production rate of cells in the presence of carbon energy source was not significantly influenced by oxygen concentration. During the batch culture cycle, the acquisition of the ability to produce HCN was preceded by oxygen reduction to growth-limiting levels. Cells which had lost the ability to produce hydrogen cyanide by oxygen treatment required protein synthesis before they could again become cyanogenic.     

Cyanide formation in preparations from Chlorella vulgaris Beijerinck: Effect of sonication and amygdalin addition

Summary A critical evaluation of a method for recovering HCN from cell extracts is presented. Since crude extracts often bind or metabolize HCN extensively, the HCN recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to HCN release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn²⁺ and peroxidase, also stimulates HCN production. In both processes, the HCN is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of HCN from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.     

Cyanogenesis by the entomopathogenic bacterium Pseudomonas entomophila

Aims:  To investigate whether the entomopathogenic bacterium Pseudomonas entomophila can synthesize hydrogen cyanide (HCN).
Methods and Results:  Cyanide production was assayed for during the growth of P. entomophila in liquid culture and during colonial growth. Pseudomonas entomophila produced HCN at a concentration of up to 40 μmol l⁻¹ during growth in liquid cultures and its production was found to be affected by oxygen availability, with levels increasing as the oxygen-transfer coefficient decreased. Pseudomonas entomophila made HCN during colonial growth at levels greater (approximately threefold) than those made by the well studied cyanogenic bacterium Pseudomonas aeruginosa .
Conclusions:  This study demonstrated unequivocally that P. entomophila can synthesize HCN, placing it among the small number of cyanogenic bacteria. Our data indicate that HCN production in P. entomophila is regulated by oxygen availability.
Significance and Impact of the Study:  Pseudomonas entomophila was recently identified to be the only pseudomonad that naturally infects and induces lethality of Drosophila melanogaster . The virulence factors which contribute to entomopathogenicity exerted by this species are largely unknown. In this study, we demonstrate that P. entomophila produces HCN, a secondary metabolite implicated in biocontrol properties and pathogenicity exerted by other bacteria.     

Cyanide formation in preparations from Chlorella and New Zealand spinach leaves: Effect of added amino acids

Summary As part of an effort to identify the natural precursor(s) of HCN in the alga Chlorella vulgaris Beijerinck, and in leaves of New Zealand spinach (Tetragonia expansa, Murr.), HCN release was measured after addition of various amino acids to illuminated algal extracts and grana preparations. Histidine is particularly effective as an HCN precursor, both with Chlorella extracts and leaf grana. With the algal extracts, d-histidine is about ten times more effective than l-histidine and histamine, whereas the two isomers (and histamine) are about equally effective with leaf grana. In the presence of leaf grana plus added Mn²⁺ and peroxidase, l-tyrosine and l-cysteine like-wise cause HCN formation; but these amino acids cause little or no HCN formation in the presence of Chlorella extracts. A stimulation of HCN production by l-histidine was observed with intact Chlorella cells. Because of the limitations of the assay method, the possibility can not be excluded that other substances than histidine may also lead to HCN generation in Chlorella vulgaris, but the results show that histidine has an important role in HCN generation by this species.Abbreviation POD peroxidase     

Relationship of Cyanogenic Capacity (HCN-c) of the Rubber Tree Hevea brasiliensis to Susceptibility to Microcyclus ulei, the Agent Causing South American Leaf Blight

Liberation of HCN from cyanogenic plant tissue depends on cyanogen content (HCN-potential), cyanogen splitting enzymes, cyanohydrin cleaving activity (hydroxynitrile lyase) and nonenzymatic cyanide detoxifying compounds. The maximal amount of HCN potentially set free is governed by the total cyanogen content, whereas the velocity of HCN liberation depends on enzymatic activities (β-glucosidase [β-G], hydroxynitrile lyase [HN]). Plants revealing a high HCN-potential and a high β-glucosidase activity generally are susceptible to infection with Microcyclus ulei. Based on the data of HCN-p and β-G activity of different Hevea species a proposal for future screening work in Hevea resistance selection is given.     

Oxygen-induced Regulation of Na/K ATPase in cerebellar granule cells

Adjustment of the Na/K ATPase activity to changes in oxygen availability is a matter of survival for neuronal cells. We have used freshly isolated rat cerebellar granule cells to study oxygen sensitivity of the Na/K ATPase function. Along with transport and hydrolytic activity of the enzyme we have monitored alterations in free radical production, cellular reduced glutathione, and ATP levels. Both active K(+) influx and ouabain-sensitive inorganic phosphate production were maximal within the physiological pO(2) range of 3-5 kPa. Transport and hydrolytic activity of the Na/K ATPase was equally suppressed under hypoxic and hyperoxic conditions. The ATPase response to changes in oxygenation was isoform specific and limited to the alpha1-containing isozyme whereas alpha2/3-containing isozymes were oxygen insensitive. Rapid activation of the enzyme within a narrow window of oxygen concentrations did not correlate with alterations in the cellular ATP content or substantial shifts in redox potential but was completely abolished when NO production by the cells was blocked by l-NAME. Taken together our observations suggest that NO and its derivatives are involved in maintenance of high Na/K ATPase activity under physiological conditions.     

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [³H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.     

Modified alternative oxidase expression results in different reactive oxygen species contents in Arabidopsis cell culture but not in whole plants

Alternative oxidase (AOX) transfers electrons from ubiquinone to oxygen in the respiratory chain of plant mitochondria. It is widely accepted that AOX functions as a mechanism decreasing the formation of reactive oxygen species (ROS) produced during respiratory electron transport. However, there are no experimental data to provide unambiguous proof of this hypothesis. We have studied growth characteristics, ROS content, and stress sensitivity in Arabidopsis transgenic lines with reduced or increased levels of AOX. We demonstrated that AOX-deficient plants grown in soil had an extended reproductive phase. Changes in AOX activity did not affect ROS content or stress sensitivity in the whole plants. However in the suspension culture, cells overexpressing AOX had significantly lower ROS content, whereas the AOX-deficient cells had higher ROS contents compared to the wild-type (WT) cells. Prooxidant treatment led to the increase in ROS content and to the reduction of viability more in the cells overexpressing AOX than in WT and AOX-deficient cells. Thus, we demonstrated that differences in the metabolism of whole plants and cultured cells might affect AOX functioning.     

Assessment of the Germination Potential of Brassica oleracea Seeds Treated with Karrikin 1 and Cyanide,Which Modify the Ethylene Biosynthetic Pathway

The relationship between ethylene and cyanide (HCN) and karrikin 1 (KAR1) in dormancy release was studied in secondary dormant Brassica oleracea L. (Chinese cabbage) seeds. Freshly harvested seeds of Brassica oleracea usually have poor germination potential. Karrikin1 (KAR1) and cyanide (HCN) are able to stimulate seed germination. However, the stimulatory effects of these two chemicals depend on the activation of the ethylene biosynthesis pathway and on ethylene perception. In this study, KAR1 and HCN application increased the activity of ethylene and of two ethylene biosynthesis enzymes, ACC synthase (ACS) and ACC oxidase (ACO). KAR1 and HCN collectively promoted the accumulation of 1 aminocyclopropane-1-carboxylic acid (ACC). In the presence of NO (nitric oxide) and KAR1, ACS and ACO activities reached their maximum levels after 36 and 42 h, respectively. Ethylene inhibitors suppressed seed germination by approximately 55%, whereas the respiratory inhibitors SHAM and NaN3 inhibited seed germination by 5–10% in the presence of HCN and KAR1. KAR1 and HCN collectively reduced the abscisic acid (ABA) content in seeds, increased the gibberellic acid (GA) content and released seed dormancy. The expression of ethylene biosynthesis genes and ethylene receptor genes (BOACO1, BOACS1, BOACS3, BOACS4, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2) provided further evidence of the involvement of ethylene in KAR1 and HCN-induced germination. BOACO1, BOACS1, BOACS5, BOACS7, BOACS9, BOACS11, BOETR1 and BOETR2 genes were up regulated in the presence of KAR1 and HCN, while the remaining genes were down regulated. The expression of various ethylene biosynthesis and ethylene receptor genes suggested functional diversification and variations in seed sensitivity in the presence of KAR1 and HCN. Therefore, in the current study, KAR1 and HCN application effectively induced the germination of B. oleracea seeds (approximately 97% germination rate) after 6 days by modifying the ethylene biosynthetic pathway.

     

Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531

We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr(531) and Tyr(554). Substituting HCN4-Tyr(531) with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr(554) with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr(531).     

Cyanogenesis of Hevea brasiliensis during Infection with Microcyclus ulei1)

Eight Hevea species have been shown to be cyanogenic. They all liberated HCN following mechanical tissue injury. Infection of Hevea leaves with conidia of the plant pathogen Microcyclus ulei leads to a large reduction of hydrocyanic acid potential, while only small amounts of HCN are set free from the leaves into the atmosphere. HCN production by infected leaves follows a reproducible pattern with a maximum between 40 and 60 hours after infection. During the entire time of infection free HCN can be detected in the leaves. From leaves of susceptible clones high amounts of HCN are liberated whereas from resistant clones only very little HCN is released. In Hevea infections with M. ulei, cyanogenesis does not lead to defense of the fungal pathogen but impairment of the resistance reaction.     

EFFECTS OF MODERATE HEAT STRESS AND DISSOLVED INORGANIC CARBON CONCENTRATION ON PHOTOSYNTHESIS AND RESPIRATION OF SYMBIODINIUM SP. (DINOPHYCEAE) IN CULTURE AND IN SYMBIOSIS1

The influence of temperature and inorganic carbon (C_i) concentration on photosynthesis was examined in whole corals and samples of cultured symbiotic dinoflagellates (Symbiodinium sp.) using combined measurements from a membrane inlet mass spectrometer and chl a fluorometer. In whole corals, O₂ production at 26°C was significantly limited at C_i concentrations below ambient seawater (～2.2 mM). Further additions of C_i up to ～10 mM caused no further stimulation of oxygenic photosynthesis. Following exposure to 30°C (2 d), net oxygen production decreased significantly in whole corals, as a result of reduced production of photosynthetically derived oxygen rather than increased host consumption. Whole corals maintained a rate of oxygen evolution around eight times lower than cultured Symbiodinium sp. at inorganic carbon concentrations <2 mM, but cultures displayed greater levels of photoinhibition following heat treatment (30°C, 2 d). Whole corals and cultured zooxanthellae differed considerably in their responses to C_i concentration and moderate heat stress, demonstrating that cultured Symbiodinium make an incongruous model for those in hospite. Reduced net oxygen evolution, in whole corals, under conditions of low C_i (<2 mM) has been interpreted in terms of possible sink limitation leading to increased nonphotochemical energy dissipation. The advantages of combined measurement of net gas exchange and fluorometry offered by this method are discussed.     

Modulation of smooth muscle phenotype in vitro by homologous cell substrate

We have developed a novel cell culturesystem that supports the shortening of smooth muscle cells. Primary ratairway smooth muscle cells were plated on an ethanol-fixed, confluentmonolayer of homologous smooth muscle cells (homologous cell substrate, HCS). Cells grown on HCS exhibited morphological and functional characteristics consistent with a differentiated phenotype. Cells onHCS were spindle shaped with a well-defined long axis, whereas cellsgrown on glass were larger and irregularly shaped. Smooth muscle-specific -actin immunostained diffusely in cells on HCS, whereas it appeared as stress fibers in cells on glass. Agonists recruited a greater fraction of HCS cells to contract, resulting ingreater changes in cell area or length on average, but the maximalcapacity of shortening of individual cells was similar between thegroups. Unlike cells on glass, cells on HCS shortened to methacholine.HCS was reversible and persisted over several passages. Agonistsstimulated intracellular Ca²⁺ oscillations in cells on HCS,whereas they elicited biphasic peak and plateau transients in cells onglass. HCS modulates smooth muscle cell phenotype in vitro.

     

Interaction of the pacemaker channel HCN1 with filamin A

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin-) indicating that interaction with filamin A affects membrane localization. Also, in filamin- cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.     

The effect of inorganic phosphate on cyanogenesis by Pseudomonas aeruginosa

The biosynthesis of hydrogen cyanide (HCN) by a strain of Pseudomonas aeruginosa is found to be significantly influenced by inorganic phosphate. Optimum HCN production occurs when the phosphate concentration is between 1 and 10 mM. Above and below this concentration the amount of HCN produced decreases sharply and at 0.1 and 100 mM phosphate low HCN production occurs. If a culture growing at 0.1 mM phosphate and producing low HCN is shifted to 10 mM phosphate, HCN biosynthesis resumes. Experiments with chloramphenicol indicate that de novo-protein synthesis is required for the process.     

Effects of parasitism and pesticide exposure on characteristics and functions of hemocyte populations in the freshwater snail Lymnaea palustris (Gastropoda, Pulmonata)

Morphological characteristics and functions of hemocytes were used to compare the immunological effects of biological and chemical stress in the freshwater snailLymnaea palustris. Animals were either infected by a trematode parasite (Metaleptocephalus sp.), or exposed to environmental contaminants, namely atrazine and hexachlorobenzene (HCB). Three populations of circulating hemocytes, morphologically and cytochemically distinct (round cells, hyalinocytes, granulocytes), were identified in both control and parasitized or pesticide-exposed snails. After 6 h of exposure, HCB and atrazine resulted in 8-fold increases in the mean total number of hemocytes, whereas only a 2.2-fold increase was observed 6 h after cercaria emission in parasitized snails. The impact of HCB was limited to the first 24 h of exposure, whereas long-lasting effects of atrazine were observed. Hyalinocytes and, to a lesser extent, round cells contributed most to the increases in hemocyte density in pesticide-exposed snails. Parasitism and atrazine treatment resulted in significant increases of lectin-stained hemocytes, whereas exposure to HCB did not affect the percentages of stained and unstained cells. Hemocyte phagocytic activity increased in HCB-exposed snails but with no concomitant change of the oxidative burst. Opposite results were obtained in atrazine-treated snail hemocytes, with unchanged phagocytosis and decreased phorbol 12-myristate 13-acetate-stimulated production of reactive oxygen intermediates. No increase in phagocytosis, or in the production of reactive oxygen intermediates, was observed in hemocytes from parasitized snails. Infection with the immunologically compatible trematode parasiteMetaleptocephalus sp. and exposure to atrazine generated similar reactions from circulating hemocytes, whereas a different response pattern was observed in HCB-exposed snails. This revised version was published online in July 2006 with corrections to the Cover Date.