Co-exposure to environmental microplastic and the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) induce distinctive alterations in the metabolome and microbial community structure in the gut of the earthworm Eisenia andrei.

Microplastics (MPs) are recognized as emergent pollutants and have become a significant environmental concern, especially when combined with other contaminants. In this study, earthworms, specifically Eisenia andrei, were exposed to MPs (at a concentration of 10 µg kg-1 of soil), herbicide 2,4-D (7 mg kg-1 of soil), and a combination of the two for 7 and 14 days. The chemical uptake in the earthworms was measured, and the bacterial and archaeal diversities in both the soil and earthworm gut were analyzed, along with the metabolomic profiles. Additionally, data integration of the two omics approaches was performed to correlate changes in gut microbial diversity and the different metabolites. Our results demonstrated that earthworms ingested MPs and increased 2,4-D accumulation. More importantly, high-throughput sequencing revealed a shift in microbial diversity depending on single or mixture exposition. Metabolomic data demonstrated an important modulation of the metabolites related to oxidative stress, inflammatory system, amino acids synthesis, energy, and nucleic acids metabolism, being more affected in case of co-exposure. Our investigation revealed the potential risks of MPs and 2,4-D herbicide combined exposure to earthworms and soil fertility, thus broadening our understanding of MPs' toxicity and impacts on terrestrial environments.

Insights on biotic and abiotic 2,4-dichlorophenoxyacetic acid degradation by anaerobic iron-cycling bacteria.

The use of the phenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been steadily increasing in recent years due to its selectivity against broad-leafed weeds and use on genetically modified crops resistant to 2,4-D. This increases the likelihood of 2,4-D persisting in agriculturally impacted soils, sediments, and aquatic systems. Aerobic microorganisms are capable of degrading 2,4-D enzymatically. Anaerobic degradation also occurs, though the enzymatic pathway is unclear. Iron-reducing bacteria (FeRB) have been hypothesized to augment anaerobic degradation through the production of a chemically reactive Fe(II) adsorbed to Fe(III) oxyhydroxides. To test whether this iron species can catalyze abiotic degradation of 2,4-D, an enrichment culture (BLA1) containing a photosynthetic Fe(II)-oxidizing bacterium (FeOB) "Candidatus Chlorobium masyuteum" and the FeRB "Candidatus Pseudopelobacter ferreus", both of which lacked known 2,4-D degradation genes was investigated. BLA1 produces Fe(II)-adsorbed to Fe(III) oxyhydroxides during alternating photoautotrophic iron oxidation and dark iron reduction (amended with acetate) cycles. No 2,4-D degradation occurred during iron oxidation by FeOB Ca. C. masyuteum or during iron reduction by FeRB Ca. P. ferreus under any incubation conditions tested (i.e., +/-Fe(II), +/-cells, and +/-light), or due to the presence of Fe(II) adsorbed to Fe(III) oxyhydroxides. Our results cast doubt on the hypothesis that the mineral-bound Fe(II) species augments the anaerobic degradation of 2,4-D in anoxic soils and waters by iron-cycling bacteria, and further justify the need to identify the genetic underpinnings of anaerobic 2,4-D degradation.

Elucidating the effects of acute and chronic exposure to 2,4-Dichlorophenoxyacetic acid on fathead minnow (Pimephales promelas) innate immunity.

Aquatic herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) formulations, are commonly used for invasive species management throughout the United States. Ecologically relevant concentrations of 2,4-D can impair essential behaviors, reduce survival, and act as an endocrine disruptor; however, there is limited knowledge of its effects on the health of non-target organisms. Here, we investigate the acute and chronic exposure impacts of 2,4-D on adult male and female fathead minnow (Pimephales promelas) innate immune function. We exposed both adult male and female fathead minnows to three different ecologically relevant concentrations of 2,4-D (0.00, 0.40, and 4.00 mg/L) and took blood samples at three acute time points (6, 24, and 96 h) and one chronic time point (30 days). We found that male fatheads had higher total white blood cell concentrations when exposed to 2,4-D at the acute time points. For the females, only proportions of specific cell types were altered when exposed to 2,4-D at the acute time points. However, we did not observe any significant impacts of chronic exposure to 2,4-D on any innate immune responses for either males or females. Overall, this study is the first step in answering an important question for game fisheries and management agencies while providing insight to future studies that investigate the impacts of herbicide exposure to freshwater fish health and immunity.

Combined toxicity of Cd and 2,4-dichlorophenoxyacetic acid on the earthworm Eisenia andrei under biochar amendment.

Due to anthropogenic activities, various pollutants can be found in agricultural soil, such as cadmium (Cd) and 2,4-dichlorophenoxyacetic acid (2,4-D). They are highly toxic and can have a negative impact on soil fertility. For remediation strategies, biochar has acquired considerable attention due to its benefits for agriculture. However, we should recognize the ecological risk posed by biochar use. In addition, little is known about its non-desirable effects on soil organisms such as earthworms, especially in the case of soil remediation. In this study, earthworms (Eisenia andrei) were exposed to soil contaminated with Cd (0.7 mg/kg), (2,4-D) (7 mg/kg), and a mixture of the two in the presence and absence of biochar (2%). A 7- and 14-day incubation experiment was carried out for this purpose. Cd and 2,4-D uptakes in earthworms' tissues, oxidative stress, cytotoxic response, DNA damage, histopathological changes, and gene expression level were assessed. Results suggested that biochar increased the bioavailability of Cd and 2,4-D and the frequency of micronuclei (MNi) and decreased the lysosomal membrane stability (LMS) in earthworms. Also, histopathological examination detected numerous alterations in animals exposed to the contaminants without any amelioration when biochar was added. The biochemical response of earthworms in terms of oxidative stress demonstrates that in the presence of biochar, animals tend to alleviate the toxicity of Cd and 2,4-D. This was also supported by transcriptomic analyses where expression gene levels related to oxidative stress were upregulated in earthworms exposed to Cd and 2,4-D + biochar. The present investigation brought new insights concerning the use of biochar in agriculture.

2,4-Dichlorophenoxyacetic acid (2,4-D) affects DNA integrity and retina structure in zebrafish larvae.

Monitoring the potential risk of herbicides in non-target organisms is a crucial issue for environmental safety. 2,4-D is an herbicide of high environmental relevance that has been shown to exert toxic effects to soil and aquatic biota. In the present study, we investigated the possible genotoxic and retinal development effects of 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide in early life stages zebrafish (Danio rerio). Genotoxicity was evaluated by measuring DNA damage using the comet assay and also by the mRNA expression of genes implicated in apoptosis and/or DNA repair. Retinal development toxicity was evaluated with histological approach. The results obtained revealed that 2,4-D alters DNA integrity of zebrafish larvae. Moreover, transcriptomic data showed a significant induction of p-53 and casp-3 genes and a significant decrease of lig-4 in larvae exposed to the highest tested concentration of 2,4-D (0.8 mg/L). This suggested that p-53 gene regulates the process of DNA repair and apoptosis with increased levels of 2,4-D. The histopathological analysis revealed that early exposure to 2,4-D damaged the structure of larvae retina. Overall, this study is the first to report the DNA damage, casp-3, lig-4 and p-53 regulation, as well as the ocular developmental toxicity in zebrafish larvae at environmentally relevant concentrations of 2,4-D herbicide.

Homeostatic regulation mechanism of spontaneous firing determines glutamate responsiveness in the midbrain dopamine neurons.

Autonomous tonic firing of the midbrain dopamine neuron is essential for maintenance of ambient dopamine level in the brain, in which intracellular Ca2+ concentration ([Ca2+]c) plays a complex but pivotal role. However, little is known about Ca2+ signals by which dopamine neurons maintain an optimum spontaneous firing rate. In the midbrain dopamine neurons, we here show that spontaneous firing evoked [Ca2+]c changes in a phasic manner in the dendritic region but a tonic manner in the soma. Tonic levels of somatic [Ca2+]c strictly tallied with spontaneous firing rates. However, manipulatory raising or lowering of [Ca2+]c with caged compounds from the resting firing state proportionally suppressed or raised spontaneous firing rate, respectively, suggesting presence of the homeostatic regulation mechanism for spontaneous firing rate via tonic [Ca2+]c changes of the soma. More importantly, abolition of this homeostatic regulation mechanism significantly exaggerated the responses of tonic firings and high-frequency phasic discharges to glutamate. Therefore, we conclude that this Ca(2+)-dependent homeostatic regulation mechanism is responsible for not only maintaining optimum rate of spontaneous firing, but also proper responses to glutamate. Perturbation of this mechanism could cause dopamine neurons to be more vulnerable to glutamate and Ca2+ toxicities.

Combined isotope and enantiomer analysis to assess the fate of phenoxy acids in a heterogeneous geologic setting at an old landfill.

Phenoxy acid herbicides and their potential metabolites represent industrial or agricultural waste that impacts groundwater and surface waters through leaching from old landfills throughout the world. Fate assessment of dichlorprop and its putative metabolite 4-CPP (2-(4-chlorophenoxy)propionic acid) is frequently obstructed by inconclusive evidence from redox conditions, heterogeneous geologic settings (e.g. clay till) and ambiguous parent-daughter relationships (i.e. 4-CPP may be daughter product or impurity of dichlorprop). For the first time, a combination of four methods was tested to assess transformation of phenoxy acids at a contaminated landfill (Risby site): analysis of (i) parent and daughter compound concentrations, (ii) enantiomer ratios (iii) compound-specific isotope analysis and (iv) enantiomer-specific isotope analysis. Additionally, water isotopes and chloride were used as conservative tracers to delineate two distinct groundwater flow paths in the clay till. Metabolite concentrations and isotope ratios of chlorinated ethenes demonstrated dechlorination activity in the area with highest leachate concentrations (hotspot) indicating favorable conditions also for dechlorination of dichlorprop to 4-CPP and further to phenoxypropionic acid. Combined evidence from concentrations, enantiomer ratios and isotope ratios of dichlorprop and 4-CPP confirmed their dechlorination in the hotspot and gave evidence for further degradation of 4-CPP downgradient of the hotspot. A combination of 4-CPP enantiomer and isotope analysis indicated different enantioselectivity and isotope fractionation, i.e. different modes of 4-CPP degradation, at different locations. This combined information was beyond the reach of any of the methods applied alone demonstrating the power of the new combined approach.

Apical Ca2+-activated potassium channels in mouse parotid acinar cells.

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

Agonist-evoked Ca(2+) wave progression requires Ca(2+) and IP(3).

Smooth muscle responds to IP(3)-generating agonists by producing Ca(2+) waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP(3) and the caged Ca(2+) buffer diazo-2. Waves, evoked by the IP(3)-generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) over a single though substantial length (approximately 30 microm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (approximately 9 microm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP(3) may explain the amplitude changes. Ca(2+) was required for IP(3)-mediated wave progression to occur. Increasing the Ca(2+) buffer capacity in a small (2 microm) region immediately in front of a CCh-evoked Ca(2+) wave halted progression at the site. However, the wave front does not progress by Ca(2+)-dependent positive feedback alone. In support, colliding [Ca(2+)](c) increases from locally released IP(3) did not annihilate but approximately doubled in amplitude. This result suggests that local IP(3)-evoked [Ca(2+)](c) increases diffused passively. Failure of local increases in IP(3) to evoke waves appears to arise from the restricted nature of the IP(3) increase. When IP(3) was elevated throughout the cell, a localized increase in Ca(2+) now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP(3) and Ca(2+) are required for active propagation of the wave front to occur.

PPARdelta ligand L-165041 ameliorates Western diet-induced hepatic lipid accumulation and inflammation in LDLR-/- mice.

Although peroxisome proliferator-activated receptor delta (PPARdelta) has been implicated in energy metabolism and lipid oxidation process, detailed roles of PPARdelta in lipid homeostasis under pathologic conditions still remain controversial. Thus, we investigated the effect of PPARdelta ligand L-165041 on Western diet-induced fatty liver using low-density lipoprotein receptor-deficient (LDLR(-/-)) mice. LDLR(-/-) mice received either L-165041 (5mg/kg/day) or vehicle (0.1N NaOH) with Western diet for 16 weeks. According to our data, L-165041 drastically reduced lipid accumulation in the liver, decreasing total hepatic cholesterol and triglyceride content compared to the vehicle group. Gene expression analysis demonstrated that L-165041 lowered hepatic expression of PPARgamma, apolipoprotein B, interleukin 1 beta (IL-1beta), and interleukin-6. In contrast, L-165041 increased hepatic expressions of PPARdelta, lipoprotein lipase (LPL), and ATP-binding cassette transporter G1 (ABCG1). Our data suggest that L-165041 might be effective in preventing Western diet-induced hepatic steatosis by regulating genes involved in lipid metabolism and the inflammatory response.

PPARdelta agonist-mediated ROS stimulates mouse embryonic stem cell proliferation through cooperation of p38 MAPK and Wnt/beta-catenin.

PPARdelta (peroxisome proliferator-activated receptor delta) is a member of the nuclear receptor superfamily. However, its function in tissues and cells is unknown, particularly as related to stem cell biology. We therefore investigated the PPARdelta effects on DNA synthesis in mouse embryonic stem cells (ES cells) and its related signal pathways. PPARdelta increased biphasic reactive oxygen species (ROS) production at 15 min and at 120 min incubation. PPARdelta significantly increased [(3)H] thymidine incorporation levels at various concentrations (10(-8) M to 10(-6) M) and incubation times (12 to 48 hr), and this activity was blocked by antioxidants. In addition, PPARdelta increased protein kinase C (PKC), cytosolic phospholipase A(2) (cPLA(2)) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, and Wnt/beta-catenin activation. PPARdelta increased the protein levels of cell cycle regulators, and these levels were abolished by antioxidants, bisindolymaleimide I, SB203580 and beta-catenin specific siRNA. In addition, the effect of PPARdelta on increased [(3)H] thymidine incorporation was blocked by bisindolymaleimide I, SB203580 and beta-catenin specific siRNA. In conclusion, PPARdelta agonist enhanced mouse ES cells proliferation through ROS-mediated p38 MAPK and Wnt/beta-catenin activation.

The role of peroxisome proliferator-activated receptor-beta/delta in epidermal growth factor-induced HaCaT cell proliferation.

Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) expression and activation is involved in the cell proliferation. However, little is known about the role of PPARbeta/delta in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPARbeta/delta mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPARbeta/delta protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPARbeta/delta binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPARbeta/delta caused selectively inhibition of PPARbeta/delta protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPARbeta/delta, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPARbeta/delta up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPARbeta/delta promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPARbeta/delta expression in a c-Jun-dependent manner and PPARbeta/delta plays a vital role in EGF-stimulated proliferation of HaCaT cells.

Selection of a support matrix for the removal of some phenoxyacetic compounds in constructed wetlands systems.

The efficiency of constructed wetlands systems in the removal of pollutants can be significantly enhanced by using a support matrix with a greater capacity to retain contaminants by sorption phenomena, ionic exchange or other physico-chemical processes. The aim of this work was to evaluate the efficiencies of 3 different materials, Light Expanded Clay Aggregates [LECA] (in two different particle sizes), Expanded Perlite and Sand, for the removal from water of one pharmaceutical compound (clofibric acid) and one pesticide (MCPA). Both belong to the class of phenoxyacetic compounds. In addition, relationships were established between the compounds' removal efficiencies and the physico-chemical properties of each material. LECA exhibited a high sorption capacity for MCPA, while the capacity for clofibric acid was more modest, but still significant. In contrast, perlite had a very limited sorption capacity while sand did not exhibit any sorption capacity for any of the compounds. LECA with smaller particle sizes showed higher efficiencies than larger grade LECA and can achieve efficiencies near 100% for the lower concentrations in the order of 1 mg l(-1). However, the use of these smaller particle media at larger scales may present problems with hydraulic conductivities. The results show that expanded clay presents important advantages in laboratory studies and could be used as a filter medium or a support matrix in constructed wetlands systems.

In vitro studies on the chemical reactivity of 2,4-dichlorophenoxyacetyl-S-acyl-CoA thioester.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a widely used broadleaf herbicide that has been associated with acute liver toxicity in exposed humans or animals. Chemically reactive metabolites of 2,4-D are proposed as mediators of 2,4-D-induced hepatotoxicity. The aim of the present study was to investigate a novel reactive metabolite of 2,4-D, namely 2,4-dichlorophenoxyacetyl-S-acyl-CoA (2,4-D-CoA), and to determine its involvement in 2,4-D covalent adduct formation. Thus, incubations of synthetic 2,4-D-CoA (106 microM) with GSH (1 mM) in phosphate buffer (pH 7.4) showed 2,4-D-CoA to be able to transacylate the cysteine sulfhydryl of GSH, resulting in the formation of 2,4-D-S-acyl-glutathione (2,4-D-SG) thioester and reaching a concentration of 65 microM after 1 h of incubation. Under similar conditions, 2,4-D-CoA was shown to covalently bind to nucleophilic groups on human serum albumin (HSA, 30 mg/ml), resulting in time-dependent 2,4-D-HSA covalent adduct formation that reached a maximum of 440 pmol/mg HSA after 1 h of incubation. In addition to these studies, incubations of [1-(14)C]2,4-D (1 mM) with rat hepatocytes showed a time-dependent covalent binding of 2,4-D to hepatocyte protein. Inhibition of acyl-CoA formation by trimethylacetic acid (2 mM) decreased the amount of covalent binding to protein in rat hepatocytes by 50%. These results indicate that 2,4-D-CoA thioester is a reactive metabolite of 2,4-D that may contribute to 2,4-D-protein adduct formation in vivo and therefore the associated hepatotoxicity.

Induction of a hypertrophic growth status of coronary smooth muscle cells is associated with an overexpression of TGF-beta.

Hypertrophy of vascular smooth muscle cells occurs during hypertension-induced remodelling of arteries and during development of arteriosclerosis and restenosis following angioplasty but the pathogenesis of the hypertrophic status is not yet fully understood. In a previous study we demonstrated that the synthetic non-sulfated, non-toxic heparin-mimicking compound RG-13577 is capable of inducing a cell cycle-arrested hypertrophic phenotype of coronary smooth muscle cells. In this study we clarify the mode of action of RG-13577 and demonstrate that the RG-13577-induced hypertrophy is associated with an increased expression of TGF-beta1 as indicated by an increase in TGF-beta1-specific protein and mRNA level. Furthermore we show that RG-13577-treated hypertrophic smooth muscle cells maintain full metabolic activity as indicated by a continuous de novo synthesis of protein and proteoglycans and that the RG-13577-induced growth arrest is caused not only by a higher expression of TGF-beta, but also by a reduced response of RG-treated cells to the mitogenic activity of bFGF, PDGF and EGF. The growth inhibitory activity of RG-13577 is reduced in the presence of neutralizing antibodies against TGF-beta. TGF-beta itself has anti-proliferative activity in serum-depleted medium. The RG-13577 effect is reversible since incubation of hypertrophic cells in RG-13577-free medium restores cell volume and [3H]thymidine incorporation to the values of untreated control cells within 4 days. We conclude, that the active metabolic status of RG-13577-treated cells in association with the overexpression of TGF-beta could promote repair processes of injured arteries after angioplasty without stimulating cell proliferation.

Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides.

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation.

A synthetic heparin-mimicking polyaromatic anionic compound RG-13577 (polymer of 4-hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr approximately 5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of (14)C-RG-13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor-related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG-13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild-type and heparan sulfate-deficient Chinese hamster ovary (CHO) cells, as well as normal- and LDL receptor negative- human skin fibroblasts bind RG-13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor-related protein (LRP) expressed a markedly reduced binding of RG-13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG-13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG-13577, suggesting that this inhibition is mediated by RG-13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and thereby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis.

Function and distribution of beta3-adrenoceptors in rat, rabbit and human urinary bladder and external urethral sphincter.

1. Activation of beta-adrenoceptors causes relaxation of the urinary bladder and contraction of the external urethral sphincter, which consists of fast-contracting skeletal muscles. A beta2-adrenoceptor agonist, clenbuterol, recently has been developed as a therapeutic drug for the treatment of urinary incontinence, however beta2-adrenoceptor agonists have undesirable effects on cardiac and striated muscle function. 2. In this study, we compared the effects of the beta2-adrenoceptor agonist, clenbuterol and of a novel beta3-adrenoceptor agonist, GS332, on urinary bladder and external urethral sphincter function in rat, rabbit and human. We also determined the distribution of beta3-adrenoceptors in human urinary bladder and external urethral sphincter, using radioligand-binding techniques. 3. Clenbuterol induced marked relaxations in rat, rabbit and human urinary bladder smooth muscles and also induced marked contractions in rat periurethral striated muscles (external urethral sphincter), while GS332 induced marked relaxations in rat and human, but not in rabbit, urinary bladder smooth muscles and induced small contractions in rat periurethral striated muscles. 4. The radioligand binding studies showed presence of beta2- and beta3-adrenoceptors in human urinary bladder, external urethral sphincter and abdominal rectus muscles. The affinities of GS332 were the highest in urinary bladder and the lowest in the skeletal (abdominal rectus) muscles, while the affinities of clenbuterol were similar in urinary bladder, external urethral sphincter and the skeletal (abdominal rectus) muscles. 5. These results suggest that GS332 could, similarly clenbuterol, have a role in the treatment of urinary frequency and urinary incontinence.

Utilization of phenoxyacetic acid, by strains using either the ortho or meta cleavage of catechol during phenol degradation, after conjugal transfer of tfdA, the gene encoding a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase.

The degradation of recalcitrant pollutants in contaminated soils and waters could be facilitated by broadening the degradative capabilities of indigenous microbes by the conjugal transfer of catabolic genes. The feasibility of establishing bacterial populations that degrade phenoxyacetic acid by conjugal transfer of tfdA, the gene encoding 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase, to phenol-degrading strains of Pseudomonas and Ralstonia was examined. The mobilizable plasmid pKJS32 served as a vector for delivery of tfdA and the regulatory gene, tfdS. Transconjugant strains that degraded phenol by an ortho cleavage of catechol grew well on phenoxyacetic acid while those employing a meta cleavage could only grow on phenoxyacetic acid in the presence of benzoic acid or after a prolonged lag period and the appearance of mutants that had gained catechol 1,2-dioxygenase activities. Thus, an ortho cleavage of catechol was essential for degradation of phenoxyacetic acid, suggesting that a product of the ortho-cleavage pathway, probably cis, cis-muconic acid, is an inducer of tfdA gene expression. Establishment of phenoxyacetic-acid-degrading soil populations by conjugal transfer of tfdA would depend on the presence of phenol-degrading recipients employing an ortho cleavage of catechol.

Characterization and regulation of catabolic genes.

Although a wide range of microorganisms have been discovered that are able to degrade highly stable, toxic xenobiotics, still many pollutants persist in the environment. Recent advances in the field of r-DNA technology has provided solutions to these problems. One important factor limiting the bioremediation of sites contaminated with certain hazardous wastes is the slow rate of degradation. This slow rate limits the practicality of using bacteria in remediating contaminated sites. It is possible to extend the range of substrates that an organism can utilize. It is even possible to endow an organism with the ability to degrade a predetermined range of xenobiotics. Because biotechnological processes are based on natural activities of microorganisms and constitute variations in classic domestic waste treatment processes, they are publicly more accepted. This is an area where genetic engineering can make a marked improvement by manipulating catabolic genes of microorganisms. Advances in r-DNA technology have opened up new avenues to move toward the goal of genetically engineered microorganisms to function as "designer biocatalysts" in which certain desirable biodegradation pathways or enzymes from different organisms are brought together in a single host with the aim of performing specific detoxification. In the last 2 decades much progress has been made in this direction, and as a result catabolic genes have been cloned and characterized for organochlorines, polychlorinated biphenyls, chlorobenzoates, naphthalene etc. The aim of this review is to provide an insight in the recent advances made on characterization and expression of catabolic genes that encode the degradation/detoxification of these persistent and toxic xenobiotic compounds.