Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.     

Aluminium Effects on ATPase Activity and Lipid Composition of Plasma Membranes in Sugar Beet Roots

The effect of aluminium (Al) in vivo and in vitro on root plasmamembranes has been studied in two sugar beet (Beta vulgarisL.) cultivars, Monohill (Al-sensitive) and Regina (relativelyAl-tolerant). Although Al in vitro inhibited the MgATPase inan uncompetitive way for both cultivars raised in the absenceof Al, the specific K⁺-activation of the MgATPase was only inhibitedby Al in cv. Monohill. Arrhenius analysis of the MgATPase activity showed that theeffect of Al in vitro depended on whether or not the plantswere exposed to Al in vivo. Al treatment in vitro of the MgATPasefrom control plants cultivated at a low pH (5·4) causedan increase in the phase transition temperature from 17 to 22°C. Only at a higher pH range (pH 6·1) could a secondtransition temperature be induced (at 9 °C). By additionof Al in vitro to plants cultivated with Al at pH 5·4,the slopes of the activity plots did not change. Aluminium changedthe K_m of the ATPase for MgATP in an opposite way by treatmentin vivo and in vitro. Lipid analyses of the plasma membranes showed that the acylcomposition differed little following Al treatment in vivo,but that the ratio of phosphatidylcholine: phosphatidylethanolamineincreased. The changes correlated with the observed change inthe K_m for the MgATPase. We conclude that the main effect ofAl on the MgATPase is not due to the formation of an Al-ATPcomplex. Instead, Al may bind to the membrane-bound enzyme(s)and/or modify the lipid environment. Key words: Aluminium, ATPase, Beta vulgaris, lipids     

Effect of Storage on the Survival and Biocontrol Activity of Pythium oligandrum in Pelleted Sugar Beet Seed

Seeds of sugar beet were pelleted with oospores of Pythium oligandrum and stored for 6 years at 8 20IC. Mycelium of P. oligandrum grew from pelleted seed when plated on cornmeal agar (CMA) within 48 h from 100% of seeds stored for 0, 2 and 4 years, and from 93% of seeds stored for 6 years. The germination of oospores removed from pelleted seed immediately after pelleting was 30% on CMA after 18 h of incubation, but storage gradually reduced germination to only 16% after 48 h of incubation for oospores removed from seed stored for 6 years. The biocontrol activity of P. oligandrum -pelleted seed was also tested after 6 years of storage in mixes of soil naturally infested with Pythium spp. and Aphanomyces cochlioides , and sand. P. oligandrum -pelleted seed had no effect in reducing damping-off due to the combined effect of Pythium spp. and A. cochlioides in 5 and 1% (v/v) soil-sand mixtures. However, in the 1% (v/v) soil-sand mixture, P. oligandrum significantly reduced Pythium spp.-induced damping-off from 33 to 26%.     

Studies on the Mode of Action of Reutericyclin

The mode of action of reutericyclin was determined with fluorescent dyes that probed the permeability of the cytoplasmic membrane by large molecules, protons, and potassium. A comparison of reutericyclin activity with those of nisin, nigericin, and valinomycin demonstrated that reutericyclin does not form pores but selectively dissipates the transmembrane proton potential.     

Studies on the Mode of Action of Polyoxins

(1) Chitin-UDP acetylglucosaminyltransferase (E.C. 2.4.1.16., chitin synthetase) in the cell-free system from phytopathogenic fungus Piricularia oryzae, and effects of various polyoxins and related compounds on the enzyme activity were studied. Polyoxins A~M, polyoxin A derivatives, polyoxin C derivatives, 5′-amino-5′-deoxyuridine, uridine and thymidine inhibited equally the incorporation of N-acetylglucosamine (GlcNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) into chitin.

(2) Competition between the above inhibitors and UDP-GlcNAc was observed by kinetic studies. The Km for UDP-GlcNAc was determined to be 3.3 × 10^?3 m and the Ki values for polyoxins A~M, except polyoxin C, were found to be in the range of 3.3 × 10^?5 m to 3.4 × 10^?6 m. For polyoxin C, 5′-amino-5′-deoxyuridine and uridine, the Ki values of 2.7 × 10^?3 m, 8.0 × 10^?3 m and 3.0 × 10^?3 m were given, respectively. The inhibitor constants for other related compounds were also calculated.

(3) The values of binding affinity, ?ΔG, for formation of substrate- or inhibitor-enzyme complexes were calculated from the Km or Ki values. In addition, partial binding affinities, ?Δg, for certain moieties or groups of polyoxins were estimated from the ?ΔG. For instance, the ?ΔG values for UDP-GlcNAc and polyoxin L were 5.7 kcal/mole and 9.2 kcal/mole, respectively. And the ?Δg values for the nucleoside moiety (part I), the carbamylpolyoxamic acid moiety (part II) and the carboxyl group at C5′ position of polyoxin L were 5.2, 3.5 and 0.7 kcal/mole, respectively.

(4) From the results obtained, the mechanism of action and relation between chemical structure and competitive inhibition of chitin synthetase were discussed.

     

Studies on the Mode of Action of Dipterex

To elucidate the reason of low mammalian toxicity of Dipterex, decomposition of this compound by rabbit tissue was investigated. Though splitting of P-C linkage was not confirmed, two glucuronides, presumably derived from the metabolite (s) of Dipterex, were isolated from the urine. Molar ratio of phosphorus and glucuronic acid in them are both estimated as 1 : 1, and they are observed to differ from trichloroethyl glucuronide. The rapid detoxification might be ascribed to the capacity of glucuronide formation as well as the enzymatic hydrolysis of DDVP, the active ingredient of Dipterex.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

Studies on the Concentration of Ramularia beticola Conidia in the Air Above Sugar Beet fields in Denmark

Concentrations of Ramularia beticola Faut. & Lamb. conidia in the air above 3 sugar beet (Beta vulgaris L.) field were registered for a period of 41 days altogether. A significant diurnal periodicity was revealed. Two periods of 3 and 5 days, respectively, included peaks of several thousands of conidia per m³ of air. The conidial concentrations were related to temperature, relative humidity (RH) and vapour pressure of the air, and to wind speed, dew point, rainfall and hours of sunshine. A significant effect of vapour pressure was observed for 2 fields. In one field with very low conidial concentrations, vapour pressure was nearly significant. In this field only wind speed significantly influenced dispersal of conidia. In an analysis of covariance including all data, 50% of the variation was explained by field, time of day, vapour pressure and number of hours of RH above 95% in the preceding 24h.     

Further Studies on Growth and Osmoregulation of Sugar Beet Leaves under Low Salinity Conditions

In previous work, Nunes and Dias (1980) demonstrated that lowsodium concentrations in the root medium of intact or decapitatedyoung sugar beet plants grown under controlled conditions modifiedleaf water relations and increased leaf area and dry weight.The present study confirms these findings and presents furtherresults concerning the effect of salt on the concentrationsof the main osmotic substrates and on the structural and chemicalfractions of the cell dry weight. Increases of water and turgor potentials (0.25 MPa and 0.4 MPa,respectively) and a small decrease in osmotic potential (0.16MPa) were found in the leaves of salt treated plants. In theseplants, osmotic potentials estimated from the concentrationof ions and organic solutes in the leaf sap agree with thosemeasured showing that the observed increase in sodium concentrationmay account for the small decrease in the osmotic potential.No changes were detected in the concentration of orthophosphateor malic acid but total acidity of the leaf sap from salt treatedplants was significantly lower. It was found that all the main components of cell dry matter(total protein, soluble sugars, pigments and crude cell wall)contributed to the dry weight increase in the salt treated plants.Among the polysaccharide fractions of the cell wall, pectinsincreased significantly relative to hemicellulose and cellulose. Key words: Sugar beet, Sodium chloride, Growth, Osmoregulation     

Studies on the Mode of Action of Organophosphorus Compounds

In order to further elucidate the mechanism of metabolic difference between sumithion and methylparathion, distribution of sumithion and methylparathion into several tissues, activation, that is, conversion into more toxic oxygen analogs, and degradation into non-toxic compounds were examined in vivo following the intravenous administration of the phosphorothioates to Guinea pigs and white rats. Sumioxon and methylparaoxon were detected in all tissues tested, among which lung and liver were richest in them. More sumioxon than methylparaoxon was found. Chese organophosphorus compounds were found to be decomposed to non-toxic desmethyl compounds and dimethyl phosphorothioic acid mainly in liver and kidney. From these results it seems improbable that the lower toxicity of sumithion than that of methylparathion results from the different in their rate of metabolism.     

Studies on the Mode of Action of Organophosphorus Compounds

For the purpose of distinguihsing sumithion from methylparathion in the mammalian metabolism, phosphorus³² labeled compounds were administered to Guinea pig and white rat. Both compounds were found to be absorbed readily, and phosphorus containing metabolites excreted chiefly into urine. By chromatographic separation and identification of the metabolites, the decomposition of sumithion was observed to proceed presumably more easily than methylparathion. From these results lower toxicity of the former toward mammals than the latter was discussed. In addition, compounds remaining in rice plant and German cockroach were also analysed.     

Studies on the Mode of Action of Polyoxin D

In the previous papers we reported that the antibiotic Polyoxin D induced the characteristic swelling of the mycelia of fungi,^1,2) and strongly inhibited the incorporation of ¹⁴C-glucosamine into the fungal cell wall chitin.³⁾ The present work has been conducted to further investigate the influence of this antibiotic on the fungal cell wall biosynthesis.

Polyoxin D did not inhibit the incorporation of ¹⁴C-glucose, ¹⁴C-amino acids and ¹⁴C-sodium acetate into the cell wall. In addition, UDP-N-acetylglucosamine, a precurcor of chitin biosynthesis of cell wall, was accumulated in the Polyoxin D-treated mycelia of Cochliobolus miyabeanus more than 150 to 160% of that accumulated in the untreated one.

Chitin synthetase prepared from Piricularia oryzae which is not treated with Polyoxin D was completely inhibited by the addition of 0.1 ppm of Polyoxin D. The fungitoxicity of Polyoxins A to G was positively related to their inhibition of ¹⁴C-glucosamine incorporation into the cell wall chitin of C. miyabeanus. From above results, it became evident that the antibiotic Polyoxin complex inhibited the biosynthesis of fungal cell wall chitin.     

Studies on the Mode of Action of Organophosphorus Compounds

It was presumed in the previous paper that sumithion was easily absorbed from the gastrointestestinal tract, similarly to methylparathion, following the oral administration to mammals. Inhibition of blood and brain cholinesterase of Guinea pigs and white rats in vivo after oral treatment with sumithion and methylparathion was determined. Inhibition of the enzymes was found to proceed rather rapidly and in most cases it reached maximum within one hour. Sumithion hindered the brain cholinesterase activity far less effectively than methylparathion. Intravenous administration of these phosphorothioates and their oxygen analogs clarified that the latters were more inhibitory on the cholinesterases and that of the two oxygen analogs, sumioxon was less effective. The poor susceptibility of the brain enzyme to sumithion was also observed in this case. This paper deals with the experimental results and their possible implications in the toxicity of the organophosphorus compounds toward mammals.     

Maize Root Phytase (Purification,Characterization, and Localization of Enzyme Activity and Its Putative Substrate)

Three phytase (EC 3.1.3.26) isoforms from the roots of 8-d-old maize (Zea mays L. var Consul) seedlings were separated from phosphatases and purified to near homogeneity. The molecular mass of the native protein was 71 kD, and the isoelectric points of the three isoforms were pH 5.0, 4.9, and 4.8. Each of the three isoforms consisted of two subunits with a molecular mass of 38 kD. The temperature and pH optima (40[deg]C, pH 5.0) of these three isoforms, as well as the apparent Michaelis constants for sodium inositol hexakisphosphate (phytate) (43, 25, and 24 [mu]M) as determined by the release of inorganic phosphate, were only slightly different. Phytate concentrations higher than 300 [mu]M were inhibitory to all three isoforms. In contrast, the dephosphorylation of 4-nitrophenyl phosphate was not inhibited by any substrate concentration, but the Michaelis constants for this substrate were considerably higher (137-157 [mu]M). Hydrolysis of phytate by the phytase isoforms is a nonrandom reaction. D/L-Inositol-1,2,3,4,5- pentakisphosphate was identified as the first and D/L-inositol-1,2,5,6-tetrakisphosphate as the second intermediate in phytate hydrolysis. Phytase activity was localized in root slices. Although phosphatase activity was present in the stele and the cortex of the primary root, phytase activity was confined to the endodermis. Phytate was identified as the putative native substrate in maize roots (45 [mu]g P g-1 dry matter). It was readily labeled upon supplying [32P]phosphate to the roots.     

Microbial Decomposition in Aquatic Environments: Combined Process of Extracellular Enzyme Activity and Substrate Uptake

The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [H]leucine as a marker for the leucine pool and l-leucine-4 methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. It was found that turnover rates of leucine (T(r), percent per hour) and hydrolysis rates of Leu-MCA (H(r), percent per hour), as well as the quotient T(r)/H(r), reflect the impact of environmental conditions on decomposition processes at both sampling sites. The quotient T(r)/H(r) is interpreted as an indirect measurement of the pool size ratio (polymers/monomers), which may serve as an index of hydrolysis-uptake coupling in bacterial utilization of dissolved protein. Calculated on an annual average basis, turnover rates are ca. nine times higher than hydrolysis rates at the polluted station and ca. five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.