Peptide conjugates of benzene and toluene metabolites in English ryegrass

Biotransformation of [1-6-¹⁴C]benzene and [1-¹⁴C]toluene in English ryegrass (Lolium perenne L.) seedlings was investigated. Vapors of these compounds were absorbed by the leaves of this plant. Benzene and toluene were oxidized, forming phenol and benzoic acid, respectively. A portion of phenol and benzoic acid was bound by low-molecular-weight peptides forming conjugates. A qualitative amino acid composition of the peptides involved in the conjugation was determined. After removing plants from the atmosphere containing [1-6-¹⁴C]benzene and [1-¹⁴C]toluene, the radioactivity of the conjugates gradually decreased. This process was accompanied by the evolution of ¹⁴CO₂, indicating the breakdown of these conjugates. Radioactive compounds thus formed were oxidized, yielding carbon dioxide. A portion of phenol and benzoic acid, along with peptide conjugation, was subjected to further oxidative transformations up to disruption of the aromatic ring. By this pathway, nonvolatile carboxylic acids, such as muconic, fumaric, succinic, malic, malonic, glycolic, and glyoxylic, were formed. Using electron microscopy, a damaging effect of benzene on the cell ultrastructure of English ryegrass leaves was shown, and this toxic effect depended on the benzene concentration.     

New 111In labeling of IgG: 111In-oxine mediated chelation

Current methods of ¹¹¹In chelate conjugation labeling of antibodies expose the protein to pH 5–6 during ¹¹¹In chelation. These conditions could be detrimental if the antibody is acid labile. We have successfully labeled human IgG via the cyclic anhydride of DPTA and ¹¹¹In-oxyquinoline(oxine). Chelation was achieved at pH 6.9–8.4 and was complete within 1 min at room temperature. The chelation was sensitive to trace metal contamination on labware and in some reagents (including commercial ¹¹¹In-oxine).     

Conditions and characteristics of the early stages of the sexual interaction of Pichia amethionina var. amethionina and Pichia amethionina var. pachycereana yeast cells

Pichia amethionina varieties have different sensitivity to the acidity of the medium: P. amethionina var. amethionina has the optimum pH 3 for agglutination and 5 for conjugation; P. amethionina var. pachycereana has the optimum pH 4-6 for agglutination and 6 for conjugation. The optimum temperature for the both organisms is 24 degrees C. Under the optimum conditions, P. amethionina var. amethionina has a 98% agglutination and a 28% conjugation; for P. amethionina var. pachycereana, these values are 83 and 55%, respectively. The agglutination of the two varieties does not depend on the presence of glucose. The conjugation of P. amethionina var. pachycereana does not occur in a medium without glucose while the conjugation of P. amethionina var. amethionina is reduced twofold under these conditions. Inositol, biotin and folic acid produce positive effect on the conjugation of P. amethionina var. pachycereana, but only folic acid favours the conjugation of P. amethionina var. amethionina. No differences have been found between the varieties by staining their cells and zygotes with FITC-ConA.     

Abscission of citrus leaf explants: no correlation with naphthaleneacetic Acid conjugation in the abscission zone

The role of α-naphthaleneacetic acid (NAA) in the control of abscission in Citrus (Citrus sinensis L. Osbeck) leaf explants and its conjugation were studied in non-aged and 24-hour-aged explants. Dipping non-aged explants in 1.5 micromolar NAA for 15 minutes immediately after excision did not delay abscission whereas 150 micromolar NAA effectively delayed it. As incubation time was prolonged up to 24 hours after excision, the delaying effect of both concentrations gradually increased. In general, both concentrations did not delay abscission when applied to 24-hour-aged explants held for an additional period of up to 24 hours. The uptake and conjugation of ¹⁴C-NAA to glucose and aspartic acid were similar in petiole, abscission zone, and leaf blade of non-aged and aged tissues, for all NAA concentrations. No correlation was established between the kinetics of abscission and the rate of conjugation in the abscission zone.     

Effect of 2,4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue

Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4-dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-¹⁴C was also inhibited in the presence of 2,4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole-3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3-acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4-dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2,4-dinitrophenol.     

Direct determination of diene conjugation and deoxyribonucleic acid (DNA) oxidative damage can explain the role of DTPA-Fe2+-O2 complex in a linoleic acid-DNA system

SUMMARY

Exposure of linoleic acid to diethylenetriminepentaacetic acid (DTPA)-Fe²⁺ complexes resulted in fast diene conjugation and peroxidized products which could further react with deoxyribonucleic acid (DNA) to cause DNA oxidative damage. In this paper, we have detected diene conjugation and DNA oxidative damage in a linoleic acid-DNA model system driven by DTPA-Fe²⁺ and found that:

• 1. in air or oxygen-saturated reaction systems, addition of hydrogen peroxide resulted in a decrease in diene conjugation and double-stranded DNA content, but had no obvious effects on the formation of DNA fluorescent products;

• 2. in anoxic conditions, addition of hydrogen peroxide had no effect on the formation of diene conjugation and fluorescent products, but resulted in a decrease of double-stranded DNA content;

• 3. in the presence of DTPA, Fe³⁺ did not stimulate the formation of diene conjugation;

• 4. the formation of diene conjugation and fluorescent products was not inhibited by superoxide dismutase, catalase, sodium benzoate, sodium azide and mannitol.

However, these ‘scavengers’ increased the percentage of double-strand DNA to different degrees. α-tocopherol, but not reduced glutathione (GSH), inhibited the formation of diene conjugates. α-tocopherol and GSH both could reduce the amounts of fluorescent products and DNA strand breaks. Taken together, these data further indicate that chelator-Fe²⁺-O₂ complex, a perferryl-type oxidant, is probably an important initiator of lipid peroxidation in the linoleic acid-DNA system.     

Inhibition of conjugation in Tetrahymena pyriformis by cerulenin. Possible requirement for de novo lipid synthesis

Conjugation in Tetrahymena pyriformis is induced by the mixing of two starved complementary mating types. Addition of the antibiotic cerulenin, a specific inhibitor of de novo lipid synthesis, upon mixing of the mating types inhibited the conjugation process. The inhibition of conjugation was found to be reversible upon washing the cells.Cerulenin inhibited [¹⁴C]acetate incorporation into the lipid fraction of the cells, while it did not affect the incorporation of [³H]leucine into proteins. Analysis of the fatty acid composition of the whole cells revealed that during conjugation the ratio of saturated to unsaturated fatty acids is markedly changed. While the ratio of saturated:unsaturated fatty acids is 0.30 in unconjugated cells, it reached a value of 0.45 in conjugated cells.     

A single-step purification of rat pancreatic and salivary amylase by affinity chromatography

Optimal conditions for the conjugation of carboxyl groups on low molecular weight molecules to reactive amino groups on rabbit immunoglobulin G (IgG) using a modified carbodiimide reaction have been investigated. Reaction of [¹⁴C]hippuric acid with N-ethyl-N′-(dimethylaminopropyl) carbodiimide at pH 5 followed by adjustment to pH 8 and coupling with rabbit IgG resulted in the formation of hippuric acid-IgG conjugates with less than 10% intra- and intermolecular IgG crosslinking. More than 93% of the bonds linking hippuric acid to IgG were stable to hydroxylamine hydrolysis, indicating the peptide properties of these bonds. This two-step process permitted a defined number of hippuric acid moieties to be loaded onto a single IgG molecule and should provide a useful method for the conjugation of molecules containing carboxyl groups to amino groups on a variety of polypeptides.     

Effect of ethylene on [C]indole-3-acetic Acid metabolism in leaf tissues of woody plants

The effect of ethylene on [¹⁴C]indole-3-acetic acid (IAA) metabolism was investigated in defoliation sensitive leaf tissues of citrus (Citrus sinensis) and resistant leaf tissues of eucalyptus (Eucalyptus camaldulensis). IAA metabolites were fractionated into 80% ethanol-soluble, H₂O-soluble, NaOH-soluble, and insoluble components. In citrus, pretreatment with 25 microliters per liter ethylene for 24 hours significantly increased the amount of ethanol- and H₂O-extractable conjugates during the first hour of incubation in [¹⁴C]IAA and increased 3- to 4-fold the formation of NaOH-extractable conjugates during the entire 6-hour incubation period. However, induction of the IAA-aspartate conjugation system was inhibited by ethylene. In eucalyptus, ethylene pretreatment only slightly stimulated the formation of IAA metabolites. Increased formation of ethanol-extractable conjugates in ethylene-pretreated eucalyptus tissues was observed only after 6 hours of incubation. Chromatographic analysis indicated that the ethanol and H₂O extracts of both species contained various low molecular weight conjugates, whereas in citrus leaf tissues high molecular weight conjugates accounted for most of the greater radioactivity detected in the NaOH extracts as a result of ethylene-pretreatment. It is suggested that ethylene may reduce the level of endogenous IAA in citrus leaf tissues by stimulating IAA conjugation.     

Conjugation of the linoleic acid oxidation product, 13-oxooctadeca-9,11-dienoic acid,a bioactive endogenous substrate for mammalian glutathione transferase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1–1, A4–4), mu (M1–1, M2–2) and pi (the allelic variants P1–1/ile, and P1–1/val) classes, and a rat theta (rT2–2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with k_cat/K_m values of 8.9 mM⁻¹ s⁻¹ for GST A1–1 and 2.14 mM⁻¹ s⁻¹ for GST A4–4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM⁻¹ s⁻¹. Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.     

Specific binding of an immunoreactive and biologically active 125I-labeled N(1)acylated substance P derivative to parotid cells

A biologically active ¹²⁵I-substance P derivative (I¹²⁵-BH-substance P), prepared by conjugation of substance P with [^125I]Bolton-Hunter reagent, binds specifically to isolated rat parotid cells. The Kd is 4 nM for I-BH-substance P, 5 nM for substance P, 0.18 μM for substance P octa(4–11)peptide, and 1.6 μM for substance P [pyroglutamyl⁶]hexa(6–11)peptide. Substance P free acid and substance P penta(7–11)peptide are much weaker competitors and the C-terminal tri(9–11)peptide has no effect at 30 μM. The binding is also inhibited by 1 μM physalaemin, eledoisin and substance P methyl ester, but not by unrelated peptides. The selective inhibition of the binding by the biologically active analogs and fragments of substance P indicates that the ¹²⁵I-labeled N(1)acylated substance P derivative may interact with a substance P receptor on parotid cells.     

PARTICIPATION OF Y89 AND Y97 IN THE CONJUGATING ACTIVITY OF Drosophila melanogaster GLUTATHIONE S‐TRANSFERASE D3 (DmGSTD3)

Drosophila melanogaster glutathione S‐transferase D3 (DmGSTD3) has a shorter amino acid sequence as compared to other GSTs known in the fruit flies. This is due to the 15 amino acid N‐terminal truncation in which normally active amino acid residue is located. The work has made use of homology modeling to visualize the arrangement of amino acid side chains in the glutathione (GSH) substrate cavity. The identified amino acids were then replaced with amino acids without functional groups in the side chains and the mutants were analyzed kinetically. Homology modeling revealed that the side chains of Y89 and Y97 were shown facing toward the substrate cavity proposing their possible role in catalyzing the conjugation. Y97A and Y89A GSH gave large changes in K_m (twofold increase), V_max (fivefold reduction), and K_cat/K_m values for GSH suggesting their significant role in the conjugation reaction. The replacement at either positions has not affected the affinity of the enzyme toward 1‐chloro‐2,4‐dinitrobenzene as no significant change in values of K_max was observed. The replacement, however, had significantly reduced the catalytic efficiency of both mutants with (K_cat/K_m)^GSH and (K_cat/K_m)^CDNB of eight‐ and twofold reduction. The recombinant DmGSTD3 has shown no activity toward 1,2‐dichloro‐4‐nitrobenzene, 2,4‐hexadienal, 2,4‐heptadienal, p‐nitrobenzyl chloride, ethacrynic acid, and sulfobromophthalein. Therefore, it was evident that DmGSTD3 has made use of distal amino acids Y97 and Y89 for GSH conjugation.     

On the role of the recipient cell during conjugation in Escherichia coli

To study the role of the E. coli recipient cell in conjugation recipient cell mutants deficient in conjugation (Con^-) were isolated. Mutants specific for F-type E. coli donor cells (ConF^-) and mutants specific deficient in conjugation with I-type donor cells (ConI^-) were isolated.Both ConF^- and ConI^- mutants were blocked in stable mating pair formation. Biochemical analysis of the mutants suggests that the outer membrane protein coded by the ompA gene and LPS are important for recipient activity in F-type conjugation while LPS is important for recipient activity in I-type conjugation.     

A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.Key words: autophagy, collaboration, gel electrophoresis, membrane, primer     

A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.     

Conjugative Transfer between Escherichia coli and Leptospira spp. as a New Genetic Tool

Our understanding of leptospiral pathogenesis, which remains poorly understood, depends on reliable genetic tools for functional analysis of genes in pathogenic strains. In this study, we report the first demonstration of conjugation between Escherichia coli and Leptospira spp. by using RP4 derivative conjugative plasmids. The DNA transfer described here was due to authentic conjugation, as shown by the requirement for cell-to-cell contact and the resistance of DNA transfers to the addition of DNase I. Transposition via conjugation of a plasmid delivering Himar1 yielded frequencies ranging from 1 × 10⁻⁶ to 8.5 × 10⁻⁸ transconjugants/recipient cell in the saprophyte L. biflexa and the pathogen L. interrogans, respectively. Analysis of mutants indicated that transposition occurs randomly, and at single sites in the genome of these strains, allowing the utilization of this system to generate libraries of transposon mutants.     

Conjugation of aspartic acid with 4-bromophenylacetic acid,an auxin analogue of aspartic acid

Tobacco mesophyll protoplasts conjugate the auxins indoleacetic acid and naphthaleneacetic acid with aspartic acid very efficiently. This conjugation was found to be correlated with the toxicity of these molecules to protoplast-derived cells grown at low densities. Among a series of halogenated phenylacetic acids, 4-bromophenylacetic was toxic to cells grown at low densities although not able to stimulate proliferation at high cell densities, as opposed to indoleacetic acid and naphthaleneacetic acid. [¹⁴C-car☐yl]-4-bromophenylacetic acid was conjugated with aspartic acid by tobacco protoplasts. Although 4-bromophenylacetic acid is not an auxin, this molecule shares with auxins some of their properties.     

Abscission: movement and conjugation of auxin

A 1-hour application of indole-3-acetic acid to bean (Phaseolus vulgaris L. cv. Red Kidney) explants inhibited abscission for an 8-hour aging period. Use of indole-3-acetic acid-¹⁴C showed that the applied indole-3-acetic acid was conjugated within explant tissue and that this conjugation mechanism accounts for loss of effectiveness of indole-3-acetic acid in inhibiting abscission after 8 hours. Reapplication of indole-3-acetic acid to an explant at a later time, before the induced aging requirement was completed reinhibited abscission. 2,4-Dichlorophenoxyacetic acid, which is not destroyed or conjugated by this system, did not lose its ability to inhibit abscission. It was concluded that indole-3-acetic acid destruction is one of the processes involved in the aging stage of abscission in explants.     

Microplate assay for aptamer-based thrombin detection using a DNA-enzyme conjugate based on histidine-tag chemistry

We report a method to prepare a DNA–enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K_d was approximately 10^?6 (M^?1) toward a His-tag present on a recombinant protein via the complexation of Ni²⁺. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni²⁺ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni²⁺ complex. The DNA–enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized.     

Auxin metabolism in representative land PLANTS

The plant hormone auxin (indole-3-acetic acid, IAA) appears to control many plant developmental processes, and studies performed in seed plants suggest that IAA conjugation is the critical mechanism to regulate free IAA concentration. The purpose of this investigation is to characterize the biochemical ability of one charophyte and 23 land plants ranging from liverworts to angiosperms to produce IAA conjugates, and to study the complexity of their conjugation patterns. Actively growing tissue was incubated with ¹⁴C-IAA, after which labeled IAA conjugates were separated using thin-layer chromatography. The conjugates were analyzed using radioimaging techniques and their tentative identity assigned by co-chromatography and/or by differential hydrolysis. The charophyte and the liverworts appear unable to conjugate IAA. The mosses and the hornwort are able to conjugate IAA into a few amide and ester conjugates. The tracheophytes examined synthesize several conjugates unique to the vascular plants, indole-3-acetyl-aspartic acid (-glutamic acid) and/or indole-3-acetyl-β-1-O-glucose, as well as a variety of other amide and ester conjugates. These three conjugation patterns are correlated to the type of conducting tissue characteristic of the plants analyzed. These biochemical differences may be indicative of significative differences in the hormonal regulation in these plant groups, thus suggesting that changes in IAA regulation accompanied the major evolutionary events in land plants.