Relationships among the herbicide and functional sites of acetohydroxy acid synthase from Chlorella emersonii

The properties of acetohydroxy acid synthase (AHAS, EC 4.1.3.18) from wild-type Chlorella emersonii (var. Emersonii, CCAP-211/11n) and two spontaneous sulfometuron methyl (SMM)-resistant mutants were examined. The AHAS from both mutants was resistant to SMM and cross-resistant to imazapyr (IM) and the triazolopyrimidine sulfonanilide herbicide XRD-498 (TP). The more-SMM-resistant mutant had AHAS with altered catalytic parameters (K _m, specificity), but unchanged sensitivity to the feedback inhibitors valine and leucine. The second mutant enzyme was less sensitive to the feedback inhibitors, but had otherwise unchanged kinetic parameters. Inhibition-competition experiments indicated that the three herbicides (SMM, IM, TP) bind in a mutually exclusive manner, but that valine can bind simultaneously with SMM or TP. The three herbicide classes apparently bind to closely overlapping sites. We suggest that the results with C. emersonii and other organisms can all be explained if there are separate binding sites for herbicides, feedback inhibitors and substrates.Abbreviations AHAS acetohydroxy acid synthase - AL acetolactate - AHB acetohydroxybutyrate - IM imazapyr - TP triazolopyrimidine sulfonanilide herbicide XRD-498 - R enzyme specificity - SMM sulfometuron methyl This research was supported in part by the United States — Israel Binational Science Foundation (BSF), Jerusalem, Israel (Grant 86-00205) and the Fund for Basic Research, Israel Academy of Sciences.     

In vivo anti inflammatory effects of the M20 IL-1 Inhibitor: I. Effects on acute inflammatory parameters

Previous studies have described an IL-1 Inhibitor produced by a myelomonocytic line developed in our laboratory (Eur J Immunol 1986; 16: 1449). This IL-1 Inhibitor was secreted by the M20 line constitutively in addition to IL-1, from which it could be separated. We have recently shown that the M20 IL-1 Inhibitor is distinct from the IL-1ra.In vitro this factor inhibited IL-1 induced proliferative responses as well as PGE₂ secretion by IL-1 induced fibroblasts. We also showed for the first time (Lymphokine Research 1988; 7(3): 268) that an IL-1 inhibitor can reduce IL-1 induced inflammatory effects. This study describes the specific effect of the M20 IL-1 Inhibitor on IL-1 induced parameters of inflammation: fever, leukocytosis and local foot pad swelling or lymph node enlargement. Purified preparations of the IL-1 Inhibitor, when injected together with IL-1, or before the IL-1, reduced fever, leukocytosis, foot pad swelling and lymph node enlargement caused by IL-1. Similar responses were obtained by injection of IL-6 or TNF, but were unaffected by the IL-1 Inhibitor, when injected together.These results indicate that the M20 IL-1 Inhibitor acts specifically on IL-1 induced responsesin vivo. The potential importance of this factor as an anti-inflammatory and immune regulatory factor, is supported by the findings of this study.Abbreviations IL-1 Interleukin 1 - IL-6 Interleukin 6 - IL-1ra Interleukin 1 receptor antagonist - TNF tumor necrosis factor     

Ligand-induced association of the type I interferon receptor components.

Two transmembrane polypeptides, IFNAR and IFN-alpha/Beta R, were previously identified as essential components of the type I interferon (IFN) receptor, but their interrelationship and role in ligand binding were not clear. To study these issues, we stably expressed and characterized the two polypeptides in host murine cells. In human cells, native IFN-alpha/beta R is a 102-kDa protein but upon reduction only a 51-kDa protein is detected. In host murine cells human IFN-alpha/beta R was expressed as a 51-kDa protein. Host cells expressing IFN-alpha/beta R bound IFN-alpha 2 with a high affinity (Kd of 3.6 nM), whereas cells expressing IFNAR exhibited no ligand binding. Upon coexpression of IFNAR and the 51-kDa IFN-alpha/beta R, the affinity for IFN-alpha 2 was increased 10-fold, approaching that of the native receptor. We show by cross-linking that both the cloned (51-kDa) and native (102-kDa) IFN-alpha/beta R bind IFN-alpha 2 to form an intermediate product, while IFNAR associates with this product to form a ternary complex. Hence, IFNAR and IFN-alpha/beta R are components of a common type I IFN receptor, cooperating in ligand binding. Ligand-induced association of IFNAR and IFN-alpha/beta R probably triggers transmembrane signaling.     

Bacterial bioluminescence as an early indication of marine fish spoilage

Summary The relationships between different microbiological and biochemical parameters and the development of bacterial luminescence associated with the spoilage of marine fish from the Mediterranean-Sea was studied during storage at different temperatures. The bioluminescence level of the bacterial suspensions that were taken from the fish skin increased during the storage; at 20°–25°C the growth and luminescence of the luminuous bacteria correlated well with the total bacterial count while at 5°C the bacterial proliferation was not accompanied by a parallel increase in luminescence.The shift in storage temperature from 25°C to 5°C stabilized the level of the luminescence of bacterial suspension taken from the winter fish which were comprised mainly by Photobacterium phosphoreum, and caused a drop in the luminescence of bacterial suspension taken from the fish caught in the summer which were comprised mainly by Beneckea barveyi. The increase in the bioluminescence level appeared earlier than the increase in trimethylamine level and occured approximately at the same time as the increase in the hypoxanthine concentration. The potential value of the use of bacterial bioluminescence as an early indication for marine fish spoilage is discussed.     

Nitric oxide as an antioxidant.

Benzoate monohydroxy compounds, and in particular salicylate, were produced during interaction of ferrous complexes with hydrogen peroxide (Fenton reaction) in a N2 environment. These reactions were inhibited when Fe complexes were flushed, prior to the addition in the model system, by nitric oxide. Methionine oxidation to ethylene by Fenton reagents was also inhibited by nitric oxide. Myoglobin in several forms such as metmyoglobin, oxymyoglobin, and nitric oxide-myoglobin were interacted with an equimolar concentration of hydrogen peroxide. Spectra changes in the visible region and the changes in membrane (microsomes) lipid peroxidation by the accumulation of thiobarbituric acid-reactive substances (TBA-RS) were determined. The results showed that metmyoglobin and oxymyoglobin were activated by H2O2 to ferryl myoglobin, which initiates membrane lipid peroxidation; but not nitric oxide-myoglobin, which, during interaction with H2O2, did not form ferryl but metmyoglobin which only poorly affected lipid peroxidation. It is assumed that nitric oxide, liganded to ferrous complexes, acts to prevent the prooxidative reaction of these complexes with H2O2.     

Physiological implications of the substrate specificities of acetohydroxy acid synthases from varied organisms.

Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VAHB/VAL = R [2-ketobutyrate]/[pyruvate] for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R greater than 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R less than 0.1).     

Acetohydroxy acid synthase is a target for leucine containing peptide toxicity in Escherichia coli.

Acetohydroxy acid synthase from a mutant resistant to leucine-containing peptides was insensitive to leucine inhibition. It is concluded that acetohydroxy acid synthase is a target for the toxicity of the high concentrations of leucine brought into Escherichia coli K-12 by leucine-containing peptides.     

Phenology and polyploidy in annual Brachypodium species (Poaceae) along the aridity gradient in Israel

Local adaptation of plants along environmental gradients provides strong evidence for clinal evolution mediated by natural selection. Plants have developed diverse strategies to mitigate stress, for example, drought escape is a phenological strategy to avoid drought stress, while polyploidy was proposed as a genomic adaptation to stress. Polyploidy as an adaptation to aridity (an environmental parameter integrating temperature and precipitation) was previously documented in annual Brachypodium spp. (Poaceae) in the Western Mediterranean. Here, we examined whether polyploidy or phenology are associated with aridity in annual Brachypodium spp. along the aridity gradient in the Eastern Mediterranean. Using flow cytometry, we determined ploidy levels of plants from natural populations along the Israeli gradient, spanning ∼424 km from mesic Mediterranean to extreme desert climates. In a common garden we recorded time of seedling emergence, flowering and senescence. We tested whether the proportion of allotetraploids in the populations and phenological traits were associated with aridity. Contrary to a previous study in the Western Mediterranean, we found no effect of aridity on the proportion of allotetraploids and diploids within populations. Interestingly, phenology was associated with aridity: time of emergence was later, while flowering and senescence were earlier in desert plants. Our results indicate that in the Eastern Mediterranean, adaptation of Brachypodium to aridity is mediated mainly by phenology, rather than ploidy level. Therefore, we suggest that genome duplication is not the main driver of adaptation to environmental stress; rather, phenological change as a drought escape mechanism may be the major adaptation.