Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

Free Radical Enhancer Xenobiotic is an Inducer of Cataract in Rabbit

Free radical enhancers, diquat, paraquat, plumbagin and juglone were used to study the oxy radical-induced damage to the rabbit lens in vitro and in vivo. Each compound caused a 6–8 fold increase in malondialdehyde (MDA) and a 30–55% decrease in reduced glutathione of the lens in vim. These peroxidative and oxidative changes were potentiated in the presence of 100% 0., abolished by N, and prevented by desferal-Mn (III) (DF-Mn) or liposomal superoxide dismutase (LSOD) indicating the involvement of O₂^?.

Diquat injected intravitreally as a single dose (300nmole in 30μl of isotonic saline) in the right eye of a 5-wk-old Dutch belted rabbit, induced early cataract after 24–72h. The lens of the contralateral control eye injected with isotonic saline had no change. In the right eye, O₂,^? and OH -productions were significantly (P < 0.01) higher; O₂-, was about 16 fold higher in the aqueous humor and vitreous humor, and 5 fold in the lens and retina, and OH. was 35 fold higher in the aqueous humor, 2 fold in vitreous humor and 5 fold in the lens and retina as compared to the respective tissues of the control eye. Enhanced lipid peroxidation in the lens was apparent from the higher levels of MDA and formation of aminophospholipid-MDA Schiff-base conjugates.

We propose that cyclic oxidation-reduction of xenobiotics coupled to the endogenous redox systems in the eye, could generate oxy radicals in excessive amounts, triggering cataractogcnesis.     

Effect of some herbicides on the production of lysine byAzotobacter chroococcum

Summary Production of lysine byAzotobacter chroococcum strain H23 was studied in chemically-defined media amended with different concentrations of alachlor, metolachlor, 2,4-D, 2,4,5-T and 2,3,6-TBA. The presence of 5, 10, and 50g/ml of alachlor or 2,3,6-TBA significantly decreased quantitative production of lysine. However, the presence 2,4-D or 2,4,5-T at concentrations of 10 and 50g/ml enhanced the production of lysine. Quantitative production of lysine was not affected as consequence of the addition of metolachlor to the culture medium, showing that the release lysine to the culture media byA. chroococcum was not affected by that herbicide.     

A VHH That Neutralizes the Zinc Metalloproteinase Activity of Botulinum Neurotoxin Type A

The current treatment of botulism is to administer animal-derived antitoxin, which frequently causes severe adverse reactions in the recipients. In this study, a heavy chain antibody fragment (VH/V^HH) phage display library was constructed by amplification of the immunoglobulin genes of a nonimmune camel, Camelus dromedarius, using primers specific to human VH gene segments. A recombinant light chain of type A botulinum toxin, BoTxA/LC, with zinc endoprotease activity was used in phage bio-panning to select phage clones displaying BoTxA/LC-bound VH/V^HH. Soluble VH/V^HH were produced and purified from 10 VH/V^HH phagemid-transformed E. coli clones. Complementary determining regions (CDRs) and immunoglobulin frameworks (FRs) of the 10 camel VH/V^HH-deduced amino acid sequences were determined. FR2 sequences of two clones showed a hallmark of camel V^HH, i.e. (F/Y)⁴²E⁴⁹R⁵⁰(G/F)⁵². The remaining eight clones had an FR2 amino acid tetrad of conventional VH, i.e. V⁴²G⁴⁹L⁵⁰W⁵². V^HH of one clone (V^HH17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of V^HH17 matched with the 194–206 amino acid residues of BoTxA/LC, which are located near the S′1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the V^HH17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the V^HH17 should be due to the V^HH insertion into the enzymatic cleft of the toxin, which is usually inaccessible to a conventional antibody molecule. This antibody fragment warrants further development as a therapeutic agent for botulism.     

Carboxylesterase activities toward pesticide esters in crops and weeds

Proteins were extracted from maize, rice, sorghum, soybean, flax and lucerne; the weeds Abutilon theophrasti, Echinochloa crus-galli, Phalaris canariensis, Setaria faberii, Setaria viridis, Sorghum halepense and the model plant Arabidopsis thaliana and assayed for carboxylesterase activity toward a range of xenobiotics. These included the pro-herbicidal esters clodinafop-propargyl, fenoxaprop-ethyl, fenthioprop-ethyl, methyl-2,4-dichlorophenoxyacetic acid (2,4-d-methyl), bromoxynil-octanoate, the herbicide-safener cloquintocet-mexyl and the pyrethroid insecticide permethrin. Highest activities were recorded with alpha-naphthyl acetate and methylumbelliferyl acetate. Esters of p-nitrophenol were also readily hydrolysed, with turnover declining as the chain length of the acyl component increased. Activities determined with model substrates were much higher than those observed with pesticide esters and were of limited value in predicting the relative rates of hydrolysis of the crop protection agents. Substrate preferences with the herbicides were typically 2,4-d-methyl>clodinafop-propargyl>fenthioprop-ethyl, fenoxaprop-ethyl and bromoxynil-octanoate. Isoelectric focussing in conjunction with staining for esterase activity using alpha-naphthyl acetate as substrate confirmed the presence of multiple carboxylesterase isoenzymes in each plant, with major qualitative differences observed between species. The presence of serine hydrolases among the resolved isoenzymes was confirmed through their selective inhibition by the organophosphate insecticide paraoxon. Our studies identify potentially exploitable differences between crops and weeds in their ability to bioactivate herbicides by enzymic hydrolysis and also highlight the usefulness of Arabidopsis as a plant model to study xenobiotic biotransformation.     

Differences in catalytic activity between rat testicular and ovarian carbonyl reductases are due to two amino acids

The sequences of rat testis carbonyl reductase (rCR1) and rat ovary carbonyl reductase (rCR2) are 98% identical, differing only at amino acids 140, 141, 143, 235 and 238. Despite such strong sequence identity, we find that rCR1 and rCR2 have different catalytic constants for metabolism of menadione and 4-benzoyl-pyridine. Compared to rCR1, rCR2 has a 20-fold lower K(m) and 5-fold lower k(cat) towards menadione and a 7-fold lower K(m) and 7-fold lower k(cat) towards 4-benzoyl-pyridine. We constructed hybrids of rCR1 and rCR2 that were changed at either residues 140, 141 and 143 or residues 235 and 238. rCR1 with residues 140, 141 and 143 of rCR2 has similar catalytic efficiency for menadione and 4-benzoyl-pyridine as rCR1. rCR1 with Thr-235 and Glu-238 of rCR2 has the catalytic constants of rCR2, indicating that it is this part of rCR2 that contributes to its lower K(m) for menadione and 4-benzoyl-pyridine. Comparisons of three-dimensional models of rCR1 and rCR2 show how Thr-235 and Glu-238 stabilize rCR2 binding of NADPH and menadione.     

Role of gamma-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae

GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding gamma-glutamyltranspeptidase (gammaGT). This is confirmed by the absence of the corresponding activity of gammaGT in the mutant with disrupted GGT1 gene. It was shown that gammaGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a gammaGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.     

The enzymatic basis for pesticide bioremediation

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.     

MEMBRANE POTENTIAL AND ENDOGENOUS ION CURRENT OF PHYCOMYCES SPORANGIOPHORES

Glass microelectrodes were inserted into the growing zone of sporangiophores of Phycomyces blakesleeanus that had been submersed in artificial pond water. The membrane potential (inside negative) increased with increasing pH of the bathing solution from an average of ?98 mV at pH 5 up to ?131 mV at pH 7. Removal of Ca²⁺ from the medium hyperpolarized the membrane potential in the wild type, but caused a significant depolarization in the blue-light-insensitive madC mutant. KCN, diethylstilbestrol, and N,N′-dicyclohexylcarbodiimide depolarized the membrane potential in both the wild type and the madC mutant, while fusicoccin had no effect. Endogenous ion current of up to 2 μA cm^?2 was measured in the growing zone of sporangiophores with an extracellular vibrating electrode. The current density and current pattern varied with the pH of the medium. At pH 5 most sporangiophores had weak inward current along the growing zone, whereas at pH 7 most sporangiophores had strong outward current. The response of the membrane potential to specific inhibitors and the presence of an endogenous ion current indicate an electrogenic H⁺-ATPase in the plasma membrane. The results show a negative correlation between growth rate of sporangiophores growing in buffered aqueous medium and magnitude of membrane potential, as well as density of outward current. They also indicate an important role of protons in controlling the growth of Phycomyces sporangiophores.