Anaerobic degradation of phthalate isomers by methanogenic consortia.
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Three methanogenic enrichment cultures, grown on ortho-phthalate, iso-phthalate, or terephthalate were obtained from digested sewage sludge or methanogenic granular sludge. Cultures grown on one of the phthalate isomers were not capable of degrading the other phthalate isomers. All three cultures had the ability to degrade benzoate. Maximum specific growth rates (microseconds max) and biomass yields (YXtotS) of the mixed cultures were determined by using both the phthalate isomers and benzoate as substrates. Comparable values for these parameters were found for all three cultures. Values for microseconds max and YXtotS were higher for growth on benzoate compared to the phthalate isomers. Based on measured and estimated values for the microbial yield of the methanogens in the mixed culture, specific yields for the phthalate and benzoate fermenting organisms were calculated. A kinetic model, involving three microbial species, was developed to predict intermediate acetate and hydrogen accumulation and the final production of methane. Values for the ratio of the concentrations of methanogenic organisms, versus the phthalate isomer and benzoate fermenting organisms, and apparent half-saturation constants (KS) for the methanogens were calculated. By using this combination of measured and estimated parameter values, a reasonable description of intermediate accumulation and methane formation was obtained, with the initial concentration of phthalate fermenting organisms being the only variable. The energetic efficiency for growth of the fermenting organisms on the phthalate isomers was calculated to be significantly smaller than for growth on benzoate. (+info)
Expression of alpha2-adrenergic receptors in rat primary afferent neurones after peripheral nerve injury or inflammation.
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1. Immunocytochemistry with polyclonal antibodies directed against specific fragments of intracellular loops of alpha2A- and alpha2C-adrenergic receptors (alpha2A-AR, alpha2C-AR) was used to explore the possibility that expression of these receptors in dorsal root ganglion (DRG) neurones of rat alters as a result of peripheral nerve injury or localized inflammation. 2. Small numbers of neurones with positive alpha2A-AR immunoreactivity (alpha2A-AR-IR) were detected in DRG from normal animals or contralateral to nerve lesions. In contrast, after complete or partial sciatic nerve transection the numbers of ipsilateral L4 and L5 DRG somata expressing alpha2A-AR-IR sharply increased (>5-fold). There was no discernible change in the number of DRG neurones exhibiting alpha2A-AR-IR innervating a region in association with localized chemically induced inflammation. 3. After nerve injury, double labelling with Fluoro-Gold, a marker of retrograde transport from transected fibres, or by immunoreactivity for c-jun protein, an indicator of injury and regeneration, suggested that many of the neurones expressing alpha2A-AR-IR were uninjured by the sciatic lesions. 4. In general the largest proportionate increase in numbers of neurones labelled by alpha2A-AR-IR after nerve lesions appeared in the medium-large diameter range (31-40 microm), a group principally composed of cell bodies of low threshold mechanoreceptors. The number of small diameter DRG neurones labelled by alpha2A-AR-IR, a category likely to include somata of nociceptors, also increased but proportionately less. 5. Relatively few DRG neurones exhibited alpha2C-AR-IR; this population did not appear to change after either nerve lesions or inflammation. 6. These observations are considered in relation to effects of nerve injury on excitation of primary afferent neurones by sympathetic activity or adrenergic agents, sympathetically related neuropathy and reports of sprouting of sympathetic fibres in DRG. (+info)
Structural identification of sulfated tyrosine in human urine.
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A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions. (+info)
Metabolism of retinaldehyde isomers in pregnant rats: 13-cis- and all-trans-retinaldehyde, but not 9-cis-retinaldehyde, yield very similar patterns of retinoid metabolites.
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Retinaldehyde (RAL), a key intermediate in retinoid metabolism, acts as a retinoic acid (RA) precursor, but is also reduced to retinol (ROH), which can subsequently be esterified to retinyl esters, the storage form of vitamin A. Limited information is available on the metabolism of geometric isomers of RAL as well as on the transplacental distribution of their metabolites, including RA isomers. Such information would be very helpful for the assessment of the teratogenic potency of RAL isomers, as teratogenesis represents a major side effect of retinoid use in pharmacotherapy. In the present study we examined concentrations of retinoids in plasma, maternal tissues, and embryos of pregnant rats 2 h after a single oral dose (100 mg/kg body weight) of all-trans-, 13-cis-, or 9-cis-RAL on gestational day 13. The main findings of this study were the very similar patterns of retinoid metabolites (consisting of retinoids with mainly the all-trans-configuration) after administration of all-trans- and 13-cis-RAL, and the high concentrations of 9-cis-RA, 9,13-dicis-RA, and 9-cis-retinoyl-beta-D-glucuronide after dosing with 9-cis-RAL. In addition, all-trans-RA as a RAL metabolite reached the embryos to a much greater extent than any of its cis-isomers. The results are discussed in view of in vitro data on enzymes involved in the biotransformation of RAL isomers. (+info)
Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma.
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Previously we reported that lysophospholipase D in rat plasma hydrolyzes endogenous unsaturated lysophosphatidylcholines (LPCs) preferentially to saturated LPCs to lysophosphatidic acids with growth factor-like and hormone-like activities. In this study, we examined the possibility that association of LPCs with different proteins in rat plasma has an effect on the preference of lysophospholipase D for unsaturated LPCs. Large portions of various LPCs were found to be recovered in the lipoprotein-poor bottom fraction. Furthermore, the percentages of LPCs associated with albumin isolated from rat plasma were shown not to be consistent with their percentage conversions to lysophosphatidic acids by lysophospholipase D on incubation of rat plasma at 37 degrees C. These results indicate that distinct distributions of LPCs in the plasma protein fractions are not critical factors for the substrate specificity of lysophospholipase D. Experiments with Nagase analbuminemic rats suggested that albumin-LPC complexes are not necessarily required for the hydrolysis by lysophospholipase D; lipoprotein-associate LPCs appeared to be good substrates for the phospholipase. We found that both saturated and unsaturated LPCs are present mainly as 1-acyl isomers in rat plasma. This result indicates that the preference of lysophospholipase D for unsaturated LPCs is not attributable to a difference in position of the acyl group attached to the glycerol backbone of LPC. In addition, lysophospholipase D was also found to attack choline phospholipids with a long chain group and a short chain alkyl group, although their percentage hydrolyses were low. Taken altogether, these results suggest that lysophospholipase D shows higher affinities for free forms of unsaturated acyl type LPCs equilibrated with albumin-bound and lipoprotein-associated forms, than for free forms of saturated acyl type LPCs and analogs of platelet-activating factor. (+info)
Cloning and characterization of RGS9-2: a striatal-enriched alternatively spliced product of the RGS9 gene.
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Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for alpha subunits of heterotrimeric G-proteins. Previous in situ hybridization analysis of mRNAs encoding RGS3-RGS11 revealed region-specific expression patterns in rat brain. RGS9 showed a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA, here referred to as RGS9-1, was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Galphat) in rod outer segments. In the present study we identify a novel splice variant of RGS9, RGS9-2, cloned from a mouse forebrain cDNA library, which encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3' untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. Immunohistochemistry showed that RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the Gi/o-coupled mu-opioid receptor response in vitro. Characterization of a bacterial artificial chromosome genomic clone of approximately 200 kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed. (+info)
Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC).
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Our group recently cloned the electrogenic Na+-HCO-3 cotransporter (NBC) from salamander kidney and later from mammalian kidney. Here we report cloning an NBC isoform (hhNBC) from a human heart cDNA library. hhNBC is identical to human renal NBC (hkNBC), except for the amino terminus, where the first 85 amino acids in hhNBC replace the first 41 amino acids of hkNBC. About 50% of the amino acid residues in this unique amino terminus are charged, compared with approximately 22% for the corresponding 41 residues in hkNBC. Northern blot analysis, with the use of the unique 5' fragment of hhNBC as a probe, shows strong expression in pancreas and expression in heart and brain, although at much lower levels. In Xenopus oocytes expressing hhNBC, adding 1.5% CO2/10 mM HCO-3 hyperpolarizes the membrane and causes a rapid fall in intracellular pH (pHi), followed by a pHi recovery. Subsequent removal of Na+ causes a depolarization and a reduced rate of pHi recovery. Removal of Cl- from the bath does not affect the pHi recovery. The stilbene derivative DIDS (200 microM) greatly reduces the hyperpolarization caused by adding CO2/HCO-3. In oocytes expressing hkNBC, the effects of adding CO2/HCO-3 and then removing Na+ were similar to those observed in oocytes expressing hhNBC. We conclude that hhNBC is an electrogenic Na+-HCO-3 cotransporter and that hkNBC is also electrogenic. (+info)
Splicing of a retained intron within ROMK K+ channel RNA generates a novel set of isoforms in rat kidney.
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The renal outer medulla K+ channel (ROMK) family of K+ channels may constitute a major pathway for K+ secretion in the distal nephron. To date, four main isoforms of this gene have been identified in the rat that differ only in their NH2-terminal amino acids and that share a common "core exon" that determines the remaining protein sequence. Using RT-PCR, we have identified a new set of ROMK isoforms in rat kidney that are generated by the deletion of a region within the ROMK core sequence that is identifiable as a typical mammalian intron. This splicing event was shown to be reproducible in vitro by detection of deleted ROMK mRNA in Madin-Darby canine kidney (MDCK) cells stably transfected with the gene for ROMK2. Translation of the deletion variant of ROMK2 was confirmed in vitro and visualized in MDCK cells following transient transfection with an enhanced green fluorescent protein tag. The deletion in this core region is predicted to generate hydrophilic proteins that are approximately one-third of the size of native ROMK and lack membrane-spanning domains. (+info)