Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.     

The relationship between chromosomal aberrations, survival and DNA repair in tumour cell lines of differential sensitivity to X-rays and sulphur mustard

A pair of cultured rat lymphosarcoma cell lines (Yoshida) with a pronounced differential sensitivity to killing with sulphur mustard (SM), but with the same sensitivity to X-rays, was examined for chromosome damage and DNA repair replication after treatment with these agents. A pair of mouse lymphoma cell lines (L5178Y) with a differential sensitivity to X-rays was similarly investigated.SM-resistant Yoshida cells suffered much less chromosome damage than sensitive cells in spite of equal alkylation of DNA, RNA and protein in sensitive and resistant cells. The pair of Yoshida cell lines sustained the same amount of chromosome damage after X-irradiation. Much less chromosome damage was observed in the radiation-resistant lymphoma cell line than in the sensitive line after X-irradiation.No differences was found between the pairs of cell lines in their capacities for repair replication after SM or X-ray treatment.Thus, the drug and radiation resistance is accompanied by, and perhaps mediated through, a reduced amount of induced chromosome damage but is not quantitatively related to the capacity for DNA repair replication.Apart from small differences in modal chromosome numbers there are no obvious karyotype differences between the sulphur mustard-sensitive and -resistant Yoshida cells or between the radiation-sensitive and -resistant lymphoma cells.     

Effects of Acetylethylcholine Mustard on [3H]Quinuclidinyl Benzilate Binding and Acetylcholine Release in Rat Brain Synaptosomes

The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37 degrees C, AMMA reduced [3H]QNB binding with an IC50 of 9 microM. Following incubation for 5 min with 50 microM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irreversibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+-induced release of newly synthesized [3H]ACh in dose-dependent manners over a 2.5-min period. At identical 50 microM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+-induced [3H]ACh release by AMMA (10-250 microM) was blocked by 2 microM atropine during a 2.5-min release incubation, but not during a 30-min release incubation. After synaptosomes were treated with 50 microM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+-induced) was reduced. AMMA (50 microM) reduced high-affinity choline uptake and ACh synthesis by greater than 90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because they were not atropine sensitive and hemicholinium-3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at presynaptic muscarinic autoreceptors.     

Alternaria incidence in some alloplasmic lines of Indian mustard (Brassica juncea (L.) Coss.)

Summary The cytoplasmic substitution lines of Brassica juncea (L.) Coss were evaluated for their field resistance to Alternaria blight (Alternaria brassicae). The euplasmic B. juncea cv. RLM 198 had a mesothetic reaction while alloplasmic B. juncea lines with cytoplasms of B. campestris, B. chinensis, and B. japonica were highly susceptible. B. nigra cytoplasm did not have any effect on the disease reaction of the B. juncea genome. However, the alloplasmic lines with the cytoplasm of B. napus and B. carinata revealed a comparatively higher degree of resistance. The study underlined the utility of cytoplasmic manipulations in modifying the phenotypic expression of nuclear genes.     

Study of factors affecting the culture of Brassica napus L. and B. juncea Coss. mesophyll protoplasts

Various factors affecting the culture of Brassica napus and B. juncea mesophyll protoplasts were examined in order to develop suitable culture media for these species. The basic components (salts and vitamins) of culture media K3 and Kao best supported cell division and colony development in protoplast culture of both species. The addition of casamino acids to Kao's medium resulted in colony browning in B. napus genotypes. B. napus protoplasts grew well with glucose as the osmotic stabilizer, whereas B. juncea protoplasts responded better to sucrose. High NAA and low 2,4-D combinations were effective in stimulating colony growth. Colony development was rapid for a range of genotypes cultured with these recommendations in these media and plant regeneration was obtained from protoplast-derived calli in both species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-Benzylaminopurine - MES 2(N-Morpholino)ethane sulfonic acid Contribution No. 931.     

Role of nitrate in regulating germination of Sinapis arvensis L. (wild mustard)

Abstract Depending on the applied concentration, nitrate can either stimulate or inhibit germination of dormant seeds of Sinapis arvensis L. (wild mustard). Seed NO^?₃ levels that corresponded to the maximal germination frequencies recorded, ranged from 0.3 to 4.4 nmol seed^?1 for applied NO^?₃ concentrations between 2.5 and 20 mol m^?3. Germination was significantly lower in seeds containing more than 5 nmol NO^?₃. Although the presence of NO^?₃ within the seed was required to promote germination, seed NO^?₃ levels were 5 to 15 times less than levels calculated from the volume of solution taken up by the seeds. Seed NO^?₃ levels also responded in a passive fashion to the external NO₃^? concentration when the seeds were incubated on filter paper or in soil. In a representative soil containing 26 mg NO^?₃-N per kg dry weight and 8 to 16% water by weight, the amount of NO^?₃ taken up by the seeds reached inhibitory levels. Our results suggest that levels of inorganic nitrogen (NO^?₃, NH⁺₄) in managed soils may play an important role in regulating germination of dormant S. arvensis seeds.     

An Evaluation of Irreversible Inhibition of Synaptosomal High-Affinity Choline Transport by Choline Mustard Aziridinium Ion

Abstract: Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium-dependent, high-affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low-affinity transport of choline. The IC₅₀ value for high-affinity transport was 0.94 μM, compared to 29 μM for low-affinity uptake. The inhibitory action of choline mustard aziridinium ion on high-affinity transport of choline was graded with respect to time; a 12-fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high-affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high-affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 × 10^?2 s^?1for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high-affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high-affinity choline carriers.