Meir Fischer

Identification and quantification of IAA and IBA in Azospirillum brasilense-inoculated maize roots

Abstract Inoculation of maize seedlings with 107 colony forming units (cfu) of Azospirillum plant−1 significantly increased the root surface area 2 weeks after sowing as compared to non-inoculated plants. Indole-3-acetic acid (IAA) was identified by gas liquid chromotography (GLC) and gas chromatography-mass spectrometry (GC-MS) in Azospirillum tryptophan-free culture medium. The amount of IAA in the medium was calculated from the gas chromatogram by isotope dilution analysis and found to be 32–40 ng ml−1. Roots of Azospirillum-inoculated maize seedlings were found to have higher amounts of both free and bound IAA as compared to control. The amount of free IAA significantly increased in the inoculated roots 2 weeks after sowing. IAA and indole-3-butyric acid (IBA) were identified by GC-MS only in the roots of Azospirillum-inoculated seedlings 2 weeks after sowing. The significance of the higher amounts of IAA following inoculation with the bacteria is discussed.

Growth response of maize roots to Azospirillum inoculation: Effect of soil organic matter content, number of rhizosphere bacteria and timing of inoculation

Abstract Root surface area of maize seedlings was the most reliable criterion in evaluation and measurement of the growth responses of maize inoculated with Azospirillum . The optimal concentration of Azospirillum inoculum for increasing root surface areas was 10 7 colony forming units (cfu) per plant. A rhizosphere-inhabiting fluorescent Pseudomonas that was used for comparison did not cause any change in root surface area. No differences were observed when maize was inoculated either at pre-emergence, post-emergence or by combining both treatments. A high concentration of Pseudomonas (10 8 cfu g −1 sand) diminished the influence of Azospirillum on root surface area. Azospirillum did not contribute to the increase of root surface area once the organic matter mixed with sand exceeded 1% by weight.

RECOMBINANT HUMAN SUPEROXIDE DISMUTASES: PRODUCTION AND POTENTIAL THERAPEUTICAL USES

In many pathological situations, tissue damage is caused by cellular generation of superoxide free radicals (O2-). These active species are generated during post-ischemic reperfusion of organs, in hyperoxic tissue, during acute and chronic inflammation and during exposure to ionizing radiation. Exogenous superoxide dismutase (SOD) was shown to significantly prevent such damage. The genes for human cytosolic Cu/ZnSOD and mitochondrial MnSOD were cloned and introduced into an E. coli expression system. The proteins were expressed in high yields and purified to homogeneity, yielding pharmaceutical-grade materials. These enzymes were used in a variety of in vivo animal models for the demonstration of their protective effects against oxidative damage. Comparative pharmacokinetic studies in rats have revealed that the half-life of Cu/ZnSOD was 6-10 min., while that of MnSOD was 5-6 hours, thus indicating that MnSOD may be superior to Cu/ZnSOD for the treatment of chronic diseases. Indeed, MnSOD was found to be effective as an anti-inflammatory agent in the rat carrageenan induced paw edema acute inflammation model. Both enzymes were also effective in ameliorating post-irradiation damage in mice exposed to whole-body or localized chest X-ray radiation.

Adsorption of Azospirillum brasilense to corn roots

Abstract The adsorption of Azospirillum brasilense strain Cd to corn roots was measured using 32 P-labeled Azospirillum cells. Binding kinetics showed that adherence of bacteria to roots increased during the first 90 min and attained a maximum level within 4.5 h. Maximum binding occurred at pH 6.1. The presence of Ca 2+ , Mg 2+ , Na + or poly- l -lysine (a polycation) reduced binding. Bacterial adherence to roots increased with increasing bacterial concentration up to 10 9 cells ml −1 of binding mixture. At higher concentrations, binding approached saturation. The bacteria attached to the roots and the bacteria in the solution around the roots were found to be in a dynamic equilibrium. Killing bacteria or roots with heat or glutaraldehyde inhibited the attachment. High concentrations of bacteria in the binding mixture (10 9 −10 11 cells ml −1 ) resulted in binding of bacteria to the roots in aggregates. The presence of several sugars, amino acids and organic acids inhibited adsorption. Chemotaxis of Azospirillum towards corn root exudates was observed. Treating the extract with trypsin eliminated this effect. At pH 6.1, Azospirillum brasilense strain Cd adsorbed in higher numbers than Klebsiella pneumoniae, Pseudomonas flourescens and Escherichia coli . Differences in adsorption levels were also found among several strains and mutants of Azospirillum .

Expression and Reconstitution of Biologically Active Human Acetylcholinesterase from Escherichia coli

Summary1.Authentic human acetylcholinesterase (AChE) was expressed inEscherichia coli under regulation of the constitutivedeo promoter or the thermoinducibleλPL promoter.2.To facilitate expression in the prokaryotic system, recombinant human AChE (rhAChE) cDNA was modified at the N terminus by oligonucleotide substitutions in order to replace some of the GC-rich regions by AT. These modifications did not alter the amino acid sequence but resulted in ample production of the protein.3.rhAChE accumulated in the cells and reached a level of 10% of total bacterial proteins. A partially purified inactive recombinant protein was recovered from inclusion bodies.4.Active rhAChE was obtained after solubilization, folding, and oxidation, although the recovery of the active enzyme was low. A 20- to 40-fold increase in enzymatically active rhAChE was achieved by replacing Cys580 by serine.5.The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity.

Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray-ionization mass spectrometry

While non‐reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post‐inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methyl‐phosphonofluoridates, were measured by electrospray‐ionization mass spectrometry (ESI‐MS). The measured mass of the free enzyme was 64 700 Da (calculated 64 695 Da) and those of the methylphosphono‐HuAChE adducts, bearing isopropyl, isobutyl, 1,2‐dimethylpropyl and 1,2,2‐trimethylpropyl substituents, were 64 820, 64 840, 64 852 and 64 860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64 780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2‐dimethylpropyl methylphosphono‐HuAChE, the change in the molecular mass and the kinetics of non‐reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to ‘aging’ of phosphonyl‐AChE adducts.

Comparative analysis of native and cysteine-deficient HIV-1 reverse transcriptase.

To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E. coli and purified to homogeneity in large quantities. The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor species of 51,000 Da. Amino acid sequence analysis of the recombinant proteins revealed that the amino termini of the two major subunits are identical to that of the virion-derived enzyme. The two cysteinyl residues at positions 38 and 280 in the RT amino acid sequence were replaced by alanine in an attempt to elucidate the role of the sulfhydryl groups in RT enzyme activities, heterodimer formation, and intrasubunit linkage. The results reported here show that the two cysteinyls are dispensable and their absence in the amino acid sequence of the reverse transcriptase does not affect DNA polymerase or ribonuclease H enzyme activities or the formation of heterodimer structures. Furthermore, inhibitors of polymerase activity such as 3-azidothymidine triphosphate, dideoxythymidine triphosphate, and tetrahydroimidazo[4,5,1-JK][1,4]benzodiazepens (1H)-one are equally effective on the mutant containing no cysteinyl residues and the wild-type enzyme.

ESMS as a Unique Tool for the Molecular Monitoring of Reactions between HuAChE and Various OP-Agents

The molecular masses of the bacterially expressed recombinant HuAChE and its conjugates with series of alkyl methylphosphonofluoridates and with diisopropyl fluorophosphate (DFP), were measured by electrospray - ionization mass spectrometry (ESMS). The mass of HuAChE (measured as 64700 Da, calc. 64695 Da) increased, following reactions with sarin, isobutyl methyl phosphorofluoridate (IBMPF), 1, 2-dimethylpropyl methyl phosphorofluoridate (DMPF), soman and DFP, by 120, 140, 150, 160 and 160 Da respectively. These values were in excellent agreement with the calculated masses of the adducts and reflected both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of the phosphyl adducts change with time to yield a common product with molecular mass of 64780 Da, which is consistent with dealkylation of the phosphonyl moieties. By sequential ESMS measurements we were able to estimate the kinetics of evolution of the aged product of the HuAChE-soman adduct (t1/2 ≈ 50 sec, at pH 6.0). This rate is in good agreement with that determined by kinetic measurement of the development of non-reactivability under similar conditions. It is important to note that in agreement with the accepted mechanism of dealkylation, only two molecular species are evident in the sequential ESMS measurements during the aging process.