The Systematic Separation and Identification of Pesticides in the First Division

The systematic identification and determination of many kinds of pesticides were tried by a combination of column, thin-layer and gas chromatography. Pesticides in the first division,¹⁾ e.g., aldrin, DMC ethylene, CPAS, DDDS, o,p′-DDT, p,p′-DDT, heptachlor, quintozene, Telodrin and tetrasul, were separated from pesticides in the second division,¹⁾ e.g., α-BHC, β-BHC, γ-BHC, trifluralin, CPA, CNP, nitrofen, endrin, dieldrin, dicofol, etc. by column chromatography. Further, pesticides in the first division were separated from each other and determined approximately by thin-layer and gas chromatography.

The recoveries of these pesticides, except for aldrin and CPAS, were about 100%. The recoveries of aldrin and CPAS were about 70 %, due to decomposition during the procedures, or to adsorption on the adsorbent during the column chromatography. It was presumed from GC-MS data that CPAS was decomposed to DDDS, tetrasul and other compounds during the procedures.     

Degradation of chlorinated hydrocarbons by Clostridium sp. isolated from lindane-amended,flooded soil

Summary A Clostridium sp., isolated from flooded soil amended with lindane (γ-BHC), decomposed methoxychlor, γ-BHC and heptachlor in that order under anaerobic condition. During the bacterial degradation of ring-labelled C¹⁴-γ-BHC, there was a net loss of radioactivity from the reaction mixture. Release of C¹⁴O₂ during the degradation of C¹⁴-γ-BHC was negligible. Methane was not detected as an end product of γ-BHC breakdown. re]19720406     

Persistence and distribution of insecticidal seed-dressings for control of the wheat bulb fly, Leptohylemyia coarctata

Samples of plants and soil from two experimental sites, one clay loam and one peaty loam, were analysed chemically to try to explain differing results with γ-BHC and dieldrin seed-dressings applied to control wheat bulb fly on different soil types, and to suggest reasons for patchy plant stands and occasional failure to control the pest. Seed dressed with dieldrin yielded more than untreated seed on both sites, whereas seed treated with γ-BHC yielded as much as seed treated with dieldrin on the clay site, but it yielded less than untreated seed on peat. The chemical analyses showed that the poor performance of γ-BHC on the peat site could not be explained by its failure to persist, because the soil still contained considerable quantities of γ-BHC at the time of insect attack. Possible reasons may be sorption of the poison by organic matter making it less available, and deeper sowing, permitting larvae to reach the plants without prolonged contact with the insecticide. Bayer 38156 (O-ethyl S-p-tolyl ethyl phosphonodithioate) and trichloro-nate (O-ethyl O-2,4,5-trichlorophenyl ethyl phosphonothionate) persisted in soil long enough and were sufficiently toxic to wheat bulb fly to suggest that organophosphorus compounds might provide alternatives to chlorinated hydrocarbon insecticides for control of the fly. Analyses made on seeds dressed at the laboratory for the experiment showed that the amounts of insecticide on seeds were smaller than expected and that the amounts on individual seeds differed greatly. Of ten samples of seeds dressed commercially with y-BHC, three carried very little insecticide, and the variations in the other seven samples were greater than with experimentally applied dressings.     

The effects on soil fauna of insecticides tested against wireworms (Agriotes spp.) in wheat

The organophosphorus insecticides Bayer 38156 (O-ethyl S-p-tolyl ethyl phosphonodithioate), trichloronate, Stauffer N 2790 (O-ethyl S-phenyl ethyl phosphonodithioate), thionazin and fenitrothion were compared with aldrin, dieldrin and γ-BHC for their effects on soil fauna, particularly wire-worms, and on crop yields in 1964 and 1965. At 1·5 lb active ingredient/acre, none of the organophosphates had as great an effect on wireworms as an aldrin spray at 2·25 lb a.i./acre or a dieldrin seed dressing at 2·25 lb a.i./acre. Some treatments significantly increased and some significantly decreased numbers of mites and Collembola. Except for Allolobophora chlorotica in plots treated with Bayer 38156, earthworm numbers were greater in plots sprayed with Bayer 38156 or aldrin, or sown with dieldrin-dressed seeds, than in untreated plots. In May 1964, one month after sowing, untreated plots had significantly fewer plants than plots sprayed with aldrin, trichloronate or Bayer 38156, or sown with γ-BHC or dieldrin-dressed seeds, but yields from untreated plots at harvest were high for such a large wireworm population and did not differ significantly from yields of treated plots in either year. The persistence of thionazin and Bayer 38156 in treated plots was measured by a bioassay using Collembola. Bayer 38156 was detected in plots 1 month after spraying but not after 6 months. Thionazin left detectable residues 1 month after spraying in the two acid plots but not in the two alkaline plots. More frequent samples taken from thionazin-treated plots in 1965 showed a similar pattern of persistence, and laboratory tests, using soil mixed with various amounts of powdered calcium carbonate, confirmed that thionazin persisted longer in more acid soils.     

Laboratory and field tests in 1966-67 on chemical control of wireworms (Agriotes spp.)

Of sixteen compounds applied to soil in laboratory tests, azinphos-ethyl, P2188 (O,O-diethyl S-chloromethyl phosphorothiolothionate), ‘Dursban’ (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), P1973 (S-(N-methoxycarbonyl-N-methylcarbamoylmethyl) dimethyl phosphorothiolothionate), B77488 (O,O-diethylphosphorothioate O-esterwith phenylglyoxylonitrile oxime) and R42211 (O,O-diethyl O-(2-diethylamino-6-methyl-pyrimidin-4-yl) phosphorothioate) killed wireworms when first tested, but in second tests with the same soils only ‘Dursban’, P2188 and B77488 did so. Treating seeds with ‘Dyfonate’ (O-ethyl S-phenyl ethyl phosphonodithioate) or with ethion/γ-BHC mixtures killed few wireworms. Three field trials compared the organophosphorus insecticides ‘Dursban’, ‘Dyfonate’ and phorate with organochlorine standards. In trials with barley and potatoes the standard was 3 lb a.i./acre (3·36 kg/ha) of aldrin. The organophosphorus compounds increased plant stands of barley almost as much as aldrin, although they killed fewer wireworms; and they protected fewer potato tubers from wireworm damage. The third trial compared the organophosphorus compounds with 0·5 lb a.i./acre (0·56 kg/ha) γ-BHC sprayed on a site drilled with sugar beet seed dressed with dieldrin. The γ-BHC increased plant stands almost as much as did 3 lb a.i./acre of the organophosphorus insecticides, and killed as many wireworms.     

Effects of sublethal doses of pesticides on the oviposition behaviour of Encarsia formosa

Female Encarsia formosa were enclosed in artificial arenas containing twenty-five Trialeurodes vaporariorum scales on a Cellophane substrate. Either the substrate or the scales were previously treated with chemicals. The number of oviposition wound marks on the scales was used as a measure of effect on parasite behaviour. The insecticides, tetradifon and γ-BHC, had their greatest effect when applied to scales. The fungicides, benomyl and dichlofluanid, were more active on the Cellophane. Pirimicarb applied to the scales decreased oviposition, but greatly increased it when applied to the substrate. It is concluded that even sublethal amounts of pesticides may seriously impair the effectiveness of this parasite in commercial biological control.     

Alterations in histone acetylation following exposure to <Superscript>60</Superscript>Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0–2 Gy ⁶⁰Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic?+?r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24 h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0–1 Gy ⁶⁰Co γ-rays. SAHA treatment significantly decreased dic?+?r/cell and MN/cell in cells exposed to 0.5 or 1.0 Gy ⁶⁰Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by ⁶⁰Co γ-radiation.     

The control of paedogenetic cecid larvae in mushroom beds by the use of thionazin

Thionazin incorporated in the casing layer of mushroom beds was shown to be highly phytotoxic even at 5 ppm, but when incorporated in compost it caused little phytotoxicity up to 20 ppm. A rate of 8 ppm gave satisfactory control of Heteropeza pygmaea Winnertz (Dipt.: Cecidomyiidae), but was less suitable against Mycophila speyeri (Barnes), for which the use of γ-BHC in the casing should be retained. A severe infestation of M. speyeri was shown to depress the total yield of mushrooms by 59% and the saleable yield by 87%.     

Comparison of two sampling methods for domestic populations of Triatoma infestans in north-west Argentina

Abstract. In mud-and-thatch houses of Santiago del Estero Province, northwest Argentina, where no insecticides had been sprayed officially to control domestic infestations of the Chagas disease vector Triatoma infestans (Klug) (Hemiptera: Reduviidae), two knockdown (KD) insecticidal collection procedures were compared with the standard flush-out (FO) method for sampling T. infestans. Bugs were collected by FO using 0.2% tetramethrin in bedrooms of (1) 41 houses of Amama village employing 1 man-hour of capture effort per house, and (2) 19 houses of Trinidad and Mercedes villages employing 4 man-hours/house. From the same houses, 2-5 days after the manual FO collection, bugs were collected by KD indoor-spraying of deltamethrin 25mga.i./m² in Amama, or burning of one γ-HCH (=γ-BHC) fumigant tablet 3.1g of γ-isomer) per bedroom in Trinidad and Mercedes. The majority of infestations were detected by both methods, the proportion of positive houses being 81% at Amama and 95% at the other villages. Although the FO method was more sensitive, at least because it was applied first, the KD method detected infestations in 25% of houses where bugs were not found by FO. Bug densities estimated by FO or by subsequent KD in each house were significantly correlated: r = 0.795 for deltamethrin; r= 0.882 for y-HCH. Compared with FO collections of T.infestans large stages, i.e. adults plus fourth and fifth instar nymphs, the KD catch averaged 0.88x with deltamethrin and 0.57× with γ-HCH, regardless of the apparent population density of bugs per house. However, the KD method has practical advantages of speed and standardization.     

Properties of γ-Glutamyltransferase from Lentinus edodes

An enzyme preparation catalyzing p-nitroaniline release from γ-glutamyl-p-nitroanilide was obtained in a 200-fold purified state from fruit bodies of an edible mushroom, Lentinus edodes. Analysis of the final preparation by differential centrifugation revealed that the enzyme was still bound with subcellular particles. The enzyme catalyzed both the hydrolysis and transfer of the γ-glutamyl moiety from γ-glutamyl-p-nitroanilide, but exhibited essentially no activity of glutaminase, glutamine aminotransferase, glutamine synthetase or γ-glutamyl cyclotransferase. With γ-glutamyl-p-nitroanilide the activity was maximal at about pH 7.6. The enzyme activity increased with an increasing concentration of Tris-HCl buffer, but not with phosphate buffer which was inhibitory. An apparent Michaelis constant of 4 mm was obtained in 0.5 m Tris-HCl buffer at pH 7.6. S-Alkylcysteine sulfoxide served as the best glutamyl acceptor. A serine-borate mixture, pCMB, Cu²⁺, Hg²⁺ and Zn²⁺ were potent inhibitors. All the experimental results, including the insoluble nature of the enzyme, allowed us to classify the Lentinus enzyme in the family of γ-glutamyl transferase.     

Metabolic engineering of Bacillus amyloliquefaciens LL3 for enhanced poly-γ-glutamic acid synthesis

Poly-γ-glutamic acid (γ-PGA) is a biocompatible and biodegradable polypeptide with wide-ranging applications in foods, cosmetics, medicine, agriculture and wastewater treatment. Bacillus amyloliquefaciens LL3 can produce γ-PGA from sucrose that can be obtained easily from sugarcane and sugar beet. In our previous work, it was found that low intracellular glutamate concentration was the limiting factor for γ-PGA production by LL3. In this study, the γ-PGA synthesis by strain LL3 was enhanced by chromosomally engineering its glutamate metabolism-relevant networks. First, the downstream metabolic pathways were partly blocked by deleting fadR, lysC, aspB, pckA, proAB, rocG and gudB. The resulting strain NK-A6 synthesized 4.84 g l⁻¹ γ-PGA, with a 31.5% increase compared with strain LL3. Second, a strong promoter P_C2up was inserted into the upstream of icd gene, to generate strain NK-A7, which further led to a 33.5% improvement in the γ-PGA titre, achieving 6.46 g l⁻¹. The NADPH level was improved by regulating the expression of pgi and gndA. Third, metabolic evolution was carried out to generate strain NK-A9E, which showed a comparable γ-PGA titre with strain NK-A7. Finally, the srf and itu operons were deleted respectively, from the original strains NK-A7 and NK-A9E. The resulting strain NK-A11 exhibited the highest γ-PGA titre (7.53 g l⁻¹), with a 2.05-fold improvement compared with LL3. The results demonstrated that the approaches described here efficiently enhanced γ-PGA production in B. amyloliquefaciens fermentation.     

The effects of insecticides on the biting behaviour of wireworms (Agriotes spp.)

Filter-paper discs soaked in nutrient were used to study how insecticides affect the biting behaviour of wireworms. When wireworms were buried in soils treated with 3.7 ppm aldrin, Bayer (38156¹), N 2790 or thionazin they stopped biting several days or weeks before they died. Three out of ten wireworms slowly recovered their biting ability after being buried for 4 days in soil containing 3.7 ppm -BHC. Biting behaviour was little affected when wireworms were confined to soils containing insecticides known to be not very toxic to wireworms.Nutrient discs treated with thionazin or -BHC were bitten less often than discs containing nutrient alone. Those treated with nutrient and aldrin were bitten almost as often as nutrient discs but the wireworms later stopped biting and died.

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Die wirkungen von insektiziden auf das fressverhalten von drahtwürmern (Agriotes spp.)

Zusammenfassung Um den Einfluß von Insektiziden auf das Freßverhalten von Drahtwürmern zu prüfen, wurden auf zweierlei Weise Filtrierpapierscheiben benutzt, die mit dem Nährstoff vollgesogen waren: a) durch Zumischen der Insektizide in den Boden, so daß die Drahtwürmer ihrer Kontaktwirkung nicht entgehen konnten (Bodenbehandlung), b) durch Aufbringen der Insektizide auf die Nährscheiben selbst (Scheibenbehandlung).Drahtwürmer hörten einige Tage oder Wochen vor ihrem Tode auf zu fressen, wenn sie in Böden vergraben waren, die mit 3,7 ppm Aldrin, Bayer 38156 (O-ethyl-S-p-totyl-ethyl phosphonodithioate), N 2790 (O-ethyl-S-phenyl-ethyl-phosphonodithioate) oder Thionazin behandel waren. 3 von 10 Drahtwürmern gewannen ihre Freßfähigkeit langsam wieder, nachdem sie für 4 Tage in Boden vergraben gewesen waren, der mit 3,7 ppm -BHC behandelt war. Die Freßfähigkeit wurde wenig beeinflußt, wenn die Drahtwürmer in Boden eingeschlossen wurden, der Bromophos, Dichlofenthion, Ethion oder RD 14838 (3-isopropylphenyl N-acetyl N-methylcarbamate) enthielt, von denen keines sehr giftig für Drahtwürmer ist.Wenn die Insektizide den Nahrungsscheiben zugefügt wurden, wurden die mit Thionazin oder -BHC behandelten Scheiben weniger oft befressen als diejenigen, die nur die Nahrung enthielten. Mit Nahrungsstoffen und Aldrin behandelte Scheiben wurden bereitwillig befressen, aber die Drahtwürmer hörten später zu fressen auf und starben.

     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 1. Comparison to the rat liver enzyme

The characteristics of the enzyme γ-glutamyltranspeptidase were determined in frog liver and compared to those of the rat. In Rana pipiens, tissue distribution studies indicated the order of activity to be: kidney >>> liver >> nerve > egg > lung > heart > skeletal muscle in homogenates. In the Rana pipiens relative to the Fischer 344 rat, the activity of the liver enzyme was somewhat greater (1·8-fold) and the kidney enzyme substantially less (25-fold). Frog liver γ-glutamyltranspeptidase displayed strain-dependent differences in activity with Rana pipiens and Rana sylvatica exhibiting comparable activities and Xenopus laevis exhibiting 20-fold lower activities. No influence of sex was apparent in Rana pipiens in contrast to the sex dependent differences observed in the Fischer 344 rat: ♀ : ♂ = 7:1. In homogenates and plasma membrane fractions of Rana pipiens, Xenopus laevis and the Fischer 344 rat, high, and comparable relative specific activities, were observed, 8–11, coupled with protein yields of 2·2–2·5 per cent indicating the enzyme to be plasma membrane bound and associated with the sinusoidal surface of the liver cell. Both the frog Rana pipiens and Xenopus laevis and Fischer 344 rat liver plasma membrane enzymes displayed comparable temperature-induced activation (1·51–1·74-fold) but with a peak for the frogs at 60°C and for the rat at 50°C. Both Acivicin and maleate inhibited the liver plasma membrane γ-glutamyltranspeptidase of both Rana pipiens and the Fischer 344 rat, but the frog enzyme was less sensitive (89 per cent decrease versus 97 per cent decrease) to 150 μM Acivicin and more sensitive (65 per cent decrease versus 35 per cent decrease at 150 mM maleate) to maleate. Kinetic studies indicated that the liver plasma membrane enzyme from Rana pipiens had a K_m of 0·61 mM and V_max of 55·6 nmol mg^?1 min^?1 and that from the Fischer 344 rat had a K_m of 3·57 mM and V_max of 71·4 nmol mg^?1 min^?1.     

A Simple Colorimetrie Assay for Aspartate Aminotransferase

γ-Glutamylmethylamide (γ-GMA) synthetase was detected in crude extracts of Methylophaga sp. AA-30, but neither methylamine dehydrogenase nor N-methylglutamate dehydrogenase was observed. A large amount of γ-GMA was accumulated in the cells when the growth on methanol-methylamine was inhibited with iodoacetate, but the accumulation was not observed in the cells grown on methanol-(NH₄)₂SO₄. It is thought that γ-GMA is a metabolic intermediate of the methylamine-dissimilating pathway in the bacterium. In addition, γ-GMA-dissimilating enzymes were found in methylamine-grown cells. The enzymes, which consisted of H protein and L protein, required α-ketoglutaric acid, Mg²⁺ or Mn²⁺, and ammonia as a cofactor. Although the enzyme catalyzed the formation of glutamate from γ-GMA, it did not catalyze the formation of N-methylglutamate. Consequently, in this bacterium, methylamine seems to be metabolized through a different pathway from the N-methylglutamate pathway.     

The potential use of the Penicillium chrysogenum antifungal protein PAF,the designed variant PAFopt and its γ-core peptide Pγopt in plant protection

The prevention of enormous crop losses caused by pesticide-resistant fungi is a serious challenge in agriculture. Application of alternative fungicides, such as antifungal proteins and peptides, provides a promising basis to overcome this problem; however, their direct use in fields suffers limitations, such as high cost of production, low stability, narrow antifungal spectrum and toxicity on plant or mammalian cells. Recently, we demonstrated that a Penicillium chrysogenum-based expression system provides a feasible tool for economic production of P. chrysogenum antifungal protein (PAF) and a rational designed variant (PAF^opt), in which the evolutionary conserved γ-core motif was modified to increase antifungal activity. In the present study, we report for the first time that γ-core modulation influences the antifungal spectrum and efficacy of PAF against important plant pathogenic ascomycetes, and the synthetic γ-core peptide Pγ^opt, a derivative of PAF^opt, is antifungal active against these pathogens in vitro. Finally, we proved the protective potential of PAF against Botrytis cinerea infection in tomato plant leaves. The lack of any toxic effects on mammalian cells and plant seedlings, as well as the high tolerance to harsh environmental conditions and proteolytic degradation further strengthen our concept for applicability of these proteins and peptide in agriculture.     

GENETICS OF DIELDRIN AND γ-BHC RESISTANCE IN THE HOUSEFLY

A factor for dieldrin resistance in a -BHC selected strain of Uruguayan origin was located in linkage group 4 (Hiroyoshi's numbering system) and showed approximately 40% crossover to the marker ct in F₁ females. Resistance to -BHC is partly due to the same factor. Factors on other linkage groups play an important part in resistance to -BHC, but when studied separately only the fifth linkage group showed a small effect. It is concluded that several factors on different chromosomes must contribute to the total resistance to -BHC.

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Genetik der dieldrin- und -cyclohexan bei der stubenfliege

Zusammenfassung Ein Faktor für Dieldrin-Resistenz in einem -Cyclohexan-selektierten Stamm uruguayischer Herkunft ist in der Koppelungsgruppe 4 (Hiroyoshi's Zählungssystem) gelegen und zeigt bei F₁-Weibchen annähernd 40% Crossing-over gegenüber dem Markierungsgen ct. Die Resistenz gegenüber -Cyclohexan beruht teilweise auf dem gleichen Faktor. Faktoren in anderen Koppelungsgruppen spielen eine wichtige Rolle bei der -Cyclohexan-Resistenz, aber bei getrennter Untersuchung zeigte nur die 5. Koppelungsgruppe eine schwache Wirkung. Es wird daraus geschlossen, daß an der Gesamtresistenz unterschiedliche Faktoren auf verschiedenen Chromosomen beteiligt sein müssen.

     

Neutrophil elastase treated dendritic cells promote the generation of CD4(+)FOXP3(+) regulatory T cells in vitro

We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-β secreting cells and reduces its allostimulatory ability. Since TGF-β has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4⁺FOXP3⁺ Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-β and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4⁺FOXP3⁺ cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4⁺FOXP3⁺ T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-β1 blocking antibody was added during the MLR culture. The increased number of CD4⁺ that express FOXP3 was also seen when CD4⁺CD25^- purified T cells were cocultured with the TGF-β producing DCs. Furthermore, these FOXP3⁺ T cells showed suppressive activity in vitro.These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.     

The Amount of γ-Amino Butyric Acid and the Activity of Glutamic Decarboxylase in Aging Leaves

The amount of γ-amino butyric acid as measured by thin-layer chromatography, and the specific activity of glutamic decarboxylase as measured both by Warburgapparatus (CO₂) and by thin-layer chromatography (γ-ABA) were determined in leaves of different ages. Datura suaveolens, Medicago sativa and Salvinia natans were used as experimental plants. The amount of γ-ABA increases in the leaves of Salvinia natans, Medicago sativa and Datura suaveolens in proportion to the age of the leaves. In the case of Salvinia natans, the amount of γ-ABA in old leaves is about twice as much as in young ones: in the case of Medicago sativa and Datura suaveolens the increase is slightly less. In Salvinia natans the amount of γ-ABA reaches its greatest value in the third to fifth leaf, in Medicago sativa and Datura suaveolens later. This increase in the amount of γ-ABA with respect to the age variation of the leaves, is reflected in the growth of leaves. In Salvinia natans the third or fourth leaf is full-grown, in Medicago sativa and Datura suaveolens this stage is reached in the seventh or eighth leaf. The activity of glutamic decarboxylase parallels the γ-ABA accumulation in that it increases when the leaves grow. The increase of glutnmic decarboxylase activity takes place more rapidly in the aging leaves of Medicago sativa than in those of Datura suaveolens. The activities observed are somewhat higher when the amounts of γ-ABA are analysed than when the CO₂ evolved is measured. K_M-values for glutamic decarboxylase were calculated using various substrate concentrations to test the enzyme activity. The K_M-values obtained were 7.0 · 10^?3M in the case of Medicago sativa, and 7.9 · 10^?3M in the case of Datura suaveolens.     

The random copolymerization of γ-benzyl-L-glutamate and L-valine N-carboxyanhydrides: Reactivity ratio and heteogeneity studies

The random copolymerization of the N-carboxyhydrides of γ-benzyl-L -glutamate and L -valine using triethylamine as the initiator in low dielectric media reults in high-molecular-weight copolymers at low convenrson. This behavior makes it possible to apply the monomer reactivity ration theory, which was dervied for addition polymerizations, and from the use of the copolymer composition equation, the respective monomer reactivity ratios, the average and incremental copolymer compositions, and the monomer feed ratio at any conversion can be determined. A comparison of the reactivity ratios for the copolymerization of γ-benzyl-L -glutamate NCA and L -valine NCA in benzene/methylene chloride (r_G = 2.1, r_V = 0.6) with those obtained using dioxane (r_G = 2.7, r_V = 0.3) indicates that the interchain compositional heterogeneity is greater for copolymers prepared in the dioxane. For Example, at 100% conversion of the monomeric NCAs, Poly[Glu(OBzl)⁵⁰Val⁵⁰] prepared in dioxance has an interchain composition ranging from 74 to 0 mol % γ-benzyl-L -glutamate, whereas in benzene/methylene chloride the interchain composition of γ-benzyl-L -glutamae ranges from 65 to 0 mol %. Once the reactivity ratios are obtained for any pair of α-amino and N-carboxyanhydrides, the use of the aforementioned parameters relating to interchain composition can give insight into the compositional heterogeneity between chains as a function of conversion and provide a basis for the preparation of random α-amino acid copolymers that are homogeneous.