Potential toxicities of HIV therapeutics in the developing infant.
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Amniotic Fluid↗
Biomedical subjects
T A Patterson
Publications and source records attributed to T A Patterson.
Transplacental pharmacokinetics and fetal distribution of 2', 3'-didehydro-3'-deoxythymidine (d4T) and its metabolites in late-term rhesus macaques.
BACKGROUND: The overall goal of human immunodeficiency virus (HIV) therapy during pregnancy is to maintain maternal health and reduce the probability of vertical transmission during gestation and delivery, while keeping toxicity risks low. Azidothymidine (AZT) is currently recommended for pregnant women infected with HIV; however, many pregnant women are unable to tolerate AZT because of toxicity. In the present study, the placental transfer and fetal accumulation of the anti-HIV compound 2',3'-didehydro-3'-deoxythymidine (d4T) and its active (triphosphorylated) and inactive (thymine and beta-aminoisobutyric acid) metabolites were examined at steady state in late-term rhesus macaques. METHODS: On the day of the hysterotomy, the mother was administered an intravenous loading dose of d4T, followed by a 3-hr steady-state intravenous infusion that also included [(3)H]d4T as a tracer. After 3 hr of infusion, the fetus was delivered by cesarean section under halothane/N(2)O anesthesia. Plasma, amniotic fluid, and tissues were analyzed for d4T and its inactive metabolites by HPLC; tissue samples were analyzed for d4T and active (phosphorylated) metabolites by strong anion-exchange HPLC. RESULTS: Maternal steady-state plasma concentrations of d4T were 1-2 microg/ml, with a fetal-to-maternal plasma ratio of 0.85 +/- 0.09. The fetal tissue distribution of radioactivity was highest in the kidney and lowest in the brain. D4T, thymine, and beta-aminoisobutyric acid were detected in all fetal tissues examined. CONCLUSIONS: Our data indicate that d4T readily crosses the placenta and is present in the fetus as parent compound or its inactive metabolites after maternal infusion. Although fetal plasma concentrations of d4T were similar to clinical d4T concentrations, no phosphorylated metabolites were detected. Teratology 62:93-99, 2000. Published 2000 Wiley-Liss, Inc.
Aminoisobutyric Acids↗
Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes.
The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.
Amino Acid Sequence↗
Incorporation of 3'-azido-3'-deoxythymidine (AZT) into fetal DNA and fetal tissue distribution of drug after infusion of pregnant late-term rhesus macaques with a human-equivalent AZT dose.
In the United States, the nucleoside analogue drug 3'-azido-3'deoxythymidine (AZT; also called zidovudine or ZDV) is given to most pregnant women who produce a positive test result for HIV-1. To investigate transplacental distribution and genotoxicity of AZT, near-term pregnant rhesus (Macaca mulatta) monkeys and their fetuses were studied. Four pregnant monkeys were continuously infused with 8 mg AZT/kg body weight for the 4 hours just prior to hysterotomy at term. This short-term AZT exposure resulted in AZT incorporation into DNA of fetal liver, lung, heart, skeletal muscle, brain, testis, and placenta, which varied between 29 and 1944 molecules of AZT/10(6) nucleotides. In contrast, values for AZT and combined metabolites, determined by radioactivity, varied between 0.94 and 5.20 microg AZT equivalents/g tissue. A fifth animal, (H076), was infused with 17.3 mg AZT/kg body weight for approximately 3 hours, followed by 1 hour without drug before hysterotomy. Similar to the 4 other monkeys, variable levels of AZT (16-147 molecules of AZT/10(6) nucleotides) were incorporated into organ DNA of H076, whereas organ tissues contained less-variable levels of AZT and metabolites (0.86-2.05 microg AZT equivalents/g tissue). For H076, at hysterotomy 1 hour after discontinuation of drug, values for AZT and the 3'-azido-3'-deoxythymidine-beta-D-glucuronide (AZTG) in fetal blood and amniotic fluid were twofold and threefold higher than those in maternal blood. Most AZT pharmacokinetic parameters in the fifth monkey were similar to those previously reported for the first 4 monkeys and those observed in a similar study of pregnant women. These data show that a short-term AZT infusion in pregnant rhesus monkeys, which have similar AZT pharmacokinetics to those present in a pregnant human, results in incorporation of drug into the DNA of placenta and most fetal organs. Data imply that the human fetus may also be subject to incorporation of AZT into DNA even after short-term AZT infusion to the mother just before delivery.
Animals↗
Characterization of wild lambdoid bacteriophages: detection of a wide distribution of phage immunity groups and identification of a nus-dependent, nonlambdoid phage group.
Temperate phages were isolated from fresh human fecal samples. Lambdoid phages were screened for growth on Nus+ but not Nus- bacteria. Approximately 100 independent lysogens of Nus-dependent phages were constructed and tested for immunity to superinfection by the same Nus-dependent phages. This identified 20 different phage immunity groups, 18 of which belonged to the lambdoid phage family. The DNA from the majority of these phages hybridized with a lambda DNA probe, and approximately 50% were recognized by anti-lambda antibodies. Furthermore most were inducible by UV light. Eleven phage recombinants with different immunity were obtained when a phage from each group was coinfected with lambda or its derivative lambdaBLK20. We also identified another immunity group with 48 members. None of these hybridized with either lambda or phi80 DNA probes nor were they recognized by anti-lambda serum. Most were not induced by UV light treatment, and no recombinants were obtained when crossed with either lambda or lambdaBLK20. Consequently, this group of Nus-dependent phages represent a new nonlambdoid phage family.
Animals↗
Kainic acid (KA)-induced seizures in Sprague-Dawley rats and the effect of dietary taurine (TAU) supplementation or deficiency.
Male Sprague-Dawley rats received TAU supplementation (1.5% in drinking water) or TAU deficient diets for 4 weeks to test for a possible neuroprotective role of TAU in KA-induced (10 mg/kg s.c.) seizures. TAU supplementation significantly increased serum and hippocampal TAU levels, but not TAU content in temporal cortex or striatum. TAU deficient diets did not attenuate serum or tissue TAU levels. Dietary TAU supplementation failed to decrease the number or latency of partial or clonic-tonic seizures or wet dog shakes, whereas a TAU deficient diet decreased the number of clonictonic and partial seizures. This study does not support previous observations of an anticonvulsant effect of TAU against KA-induced seizures. KA-treatment decreased alpha 2-adrenergic receptor binding sites and TAU content in the temporal cortex across all dietary treatment groups, supporting previous evidence of severe KA-induced damage and neuronal loss in this brain region.
Amino Acids↗
Neurotransmitter transporters: target for endocrine regulation.
Aminergic signaling in the CNS is terminated by clearance from the synapse via high-affinity transporter molecules in the presynaptic membrane. Relatively recent sequence identification of these molecules has now permitted the initiation of studies of regulation of transporter function at the cellular and systems levels. In vitro studies provide evidence that the transporters for dopamine, serotonin, and gamma-aminobutyric acid are substrates for regulation by protein kinase C signaling. In vivo studies provide evidence that insulin and adrenal and gonadal steroid hormones may regulate the synthesis and activity of the transporters. Future directions should permit evaluation of the role of endocrine regulation in neurotransmitter clearance, and thus in the maintenance of normal CNS aminergic signaling.
Animals↗
Sodium depletion and aldosterone decrease dopamine transporter activity in nucleus accumbens but not striatum.
Motivated behaviors, including sodium (Na) appetite, are correlated with increased dopamine (DA) transmission in the nucleus accumbens (NAc). DA transporter (DAT) modulation affects DA transmission and may play a role in motivated behaviors. In vivo Na depletion, which reliably induces Na appetite, was correlated with robust decreases in DA uptake via the DAT in the rat NAc with rotating disk electrode voltammetry [1,277 +/- 162 vs. 575 +/- 89 pmol. s-1. g-1; Vmax of transport for control vs. Na-depleted tissue]. Plasma aldosterone (Aldo) levels increase after in vivo Na depletion and contribute to Na appetite. Decreased DAT activity in the NAc was observed after in vitro Aldo treatment (428 +/- 28 vs. 300 +/- 25 pmol. s-1. g-1). Neither treatment affected DAT activity in the striatum. These results suggest that a direct action of Aldo is one possible mechanism by which Na depletion induces a reduction in DAT activity in the NAc. Reduced DAT activity may play a role in generating increased NAc DA transmission during Na appetite, which may underlie the motivating properties of Na for the Na-depleted rat.
Aldosterone↗
Dopamine transporter mRNA is increased in the CNS of Zucker fatty (fa/fa) rats.
The obese Zucker fa/fa rat is characterized by hyperinsulinemia, obesity, and altered monoamine metabolism in the central nervous system (CNS). It has been proposed that the changes in monoamine metabolism may contribute to the metabolic pathophysiology of these animals. Because it has been reported that insulin may regulate the catecholamine reuptake transporters, which terminate monoaminergic synaptic signaling, in the present study we tested whether messenger ribonucleic acid (mRNA) levels for the noradrenergic (NE) or dopaminergic (DA) transporters were altered in obese fa/fa vs. lean Fa/Fa Zucker rats. We found significantly elevated DA transporter levels in both the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) and zona incerta (ZI) of obese Zucker fa/fa rats (164 +/- 24% of control levels, p = .024; and 316 +/- 61% of control levels, p = .019, respectively). Measurement of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for NE and DA synthesis revealed no effect of the fa gene in either NE or DA neurons. These findings suggest that increased DA clearance, and perhaps decreased DA signaling, may occur in the obese Zucker fa/fa rat.
Animals↗
Polymorphisms in the human DNA ligase I gene (LIG1) including a complex GT repeat.
Sequencing of a human DNA ligase I cDNA clone derived from HeLa cells revealed two unreported differences with the published sequence: a single base change and a three-base deletion. Both differences are in exon 6, and were analyzed by amplifying a segment containing exon 5, intron 6, and exon 6. The first finding was that intron 6 is approximately 2.6 kb in size, not the 1 kb reported in the literature. By sequence analysis of amplified segments, the single-base difference in exon 6 was shown to be polymorphic, with HeLa cells heterozygous for the A/C difference. Analysis of 60 unrelated individuals found a frequency of 0.5 for each allele. Primer extension reactions across the exon 5/exon 6 boundary were performed on cDNA obtained from HeLa cells and human thymus. The results show that the three-base deletion is due to a variation in splicing. For both HeLa and thymus, two-thirds of the transcripts are like the published cDNA sequence and one-third have the three-base deletion. Finally, sequencing of part of intron 6 revealed the presence of a complex GT repeat consisting of a 48-50 nucleotide polypurine tract followed by a variable number of GT residues. This entire unit of polypurine tract plus GTs is repeated three times. Detection of the repeated sequences required the development of specialized cloning and PCR conditions. Analysis of a pedigree showed that this complex repeat is polymorphic.
Base Sequence↗
Food deprivation decreases mRNA and activity of the rat dopamine transporter.
We have hypothesized that the midbrain dopamine (DA) neurons are a target for insulin action in the central nervous system (CNS). In support of this hypothesis, we have previously demonstrated that direct intracerebroventricular infusion of insulin results in an increase in mRNA levels for the DA reuptake transporter (DAT). In this study, 24- to 36-hour food deprivation was used as a model of decreased CNS insulin levels, to test whether DAT mRNA levels, DAT protein concentration or DAT functional activity would be decreased. DAT mRNA levels, assessed by in situ hybridization, were significantly decreased in the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) (77 +/- 7% of controls, p < 0.05) of food-deprived (hypoinsulinemic) rats. Binding of a specific high-affinity DAT ligand (125I-RTI-121) to membranes from brain regions of fasted or free-feeding rats provided an estimate of DAT protein, which was unchanged in both of the major terminal projection fields, the striatum and nucleus accumbens (NAc). In addition, we utilized the rotating disk electrode voltametry technique to assess possible changes in the function of the DAT in fasting (hypoinsulinemic) rats. The Vmax of DA uptake was significantly decreased (87 +/- 7% of control, p < 0.05), without a change in the Km of uptake, in striatum from fasted rats. In vitro incubation with a physiological concentration (1 nM) of insulin resulted in an increase of striatal DA uptake to control levels. We conclude that striatal DAT function can be modulated by fasting and nutritional status, with a contribution by insulin.
Animals↗
Substrate binding by human apurinic/apyrimidinic endonuclease indicates a Briggs-Haldane mechanism.
Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5' to a DNA abasic site. We have characterized this reaction by steady-state and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli. The substrate was a 49-base pair oligonucleotide with an abasic site at position 21. This substrate was generated by treating a 49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase. The enzymatic products of the AP endo nicking reaction were a 20-mer with a hydroxyl group at the 3'-terminus and a 28-mer with a phosphodeoxyribose at the 5'-terminus. To obtain maximal enzymatic activity, it was necessary to stabilize the abasic site during treatment with uracil-DNA glycosylase with a reducing agent. Otherwise, a 20-mer with phosphoribose at the 3'-terminus resulted from beta-elimination. In agreement with others, Km and kcat were 100 nM and 10 s(-1), respectively. Heat treatment of the abasic site-containing 49-mer without enzyme also resulted in conversion to the beta-elimination product. The resultant heat degradation product was an efficient inhibitor of AP endo with a Ki of 30 nM. The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+. Electrophoretic mobility shift assays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations. The association and dissociation rate constants for substrate binding to AP endo were determined by using a challenge assay to follow AP endo-substrate complex formation. Heat degradation product together with heparin served as an effective trap for free enzyme. The results are consistent with a Briggs-Haldane mechanism where k(on) and k(off) are 5 x 10(7) M(-1) s(-1) and 0.04 s(-1), respectively (Kd = 0.8 nM), kcat is 10 s(-1), and product release is very rapid (i.e. k(off,product) >> 10 s(-1)). This scheme is in excellent agreement with the measured steady-state kinetic parameters.
DNA-(Apurinic or Apyrimidinic Site) Lyase↗
Agonist-induced phosphorylation of the kappa-opioid receptor.
Antipeptide antibodies against the kappa-opioid receptor were used to test whether acute or chronic exposure to kappa agonists altered the phosphorylation state of the kappa-opioid receptor. Immunoprecipitation of the kappa receptor from guinea pig hippocampal slices preincubated in [32P]orthophosphoric acid revealed a basal phosphorylation of the kappa-opioid receptor. The amount of 32P incorporation into the receptor was increased following a 75-min treatment with the kappa agonist U50,488H. This effect was blocked by the selective kappa receptor antagonist norbinaltorphimine. The time course of this change in the phosphorylation state of the receptor correlated with a desensitization of the electrophysiological response to kappa agonists measured in the dentate gyrus of hippocampal slices. The phosphorylation state of the kappa-opioid receptor was also elevated in brain slices from guinea pigs made tolerant to U50,488H by 5 days of continuous exposure and then maintained in kappa agonist to avoid acute opiate withdrawal. The results of this study show that the kappa-opioid receptor was phosphorylated in an agonist-dependent manner in brain slices taken from untreated and U50,488H-tolerant animals.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Transplacental pharmacokinetics and fetal distribution of azidothymidine, its glucuronide, and phosphorylated metabolites in late-term rhesus macaques after maternal infusion.
3'-Azido-3'-deoxythymidine (AZT) is currently prescribed to pregnant women infected with human immunodeficiency virus to reduce the risk of vertical transmission of the virus to the fetus. Consequently, more information is needed concerning the placental transfer and tissue distribution of AZT and its metabolites. In the present study, the placental transfer and fetal accumulation of AZT, its glucuronide metabolite [3'-azido-3'-deoxythymidine-beta-D-glucuronide (AZTG)], and phosphorylated metabolites were examined at steady-state in near-term rhesus macaques. One to 2 weeks before a chronic infusion, an intravenous bolus of 8 mg/kg AZT was administered to pregnant animals to determine the dose of AZT needed to reach steady-state plasma concentrations. On the day of hysterotomy, the mother was administered an intravenous loading dose of AZT, followed by a 3-hr steady-state intravenous infusion that also included a trace of [3H]AZT. After 3 hr of infusion, the mother was anesthetized, and the fetus was delivered. Plasma and amniotic fluid were analyzed for AZT and AZTG by HPLC, and tissue samples were analyzed for AZT, AZTG, and phosphorylated metabolites by strong anion exchange HPLC. Maternal steady-state plasma concentrations were 1.3-2.2 micrograms/ml for AZT and 2.3-8.0 micrograms/ml for AZTG. Fetal AZT and AZTG plasma concentrations were both lower (0.98-2.3 micrograms/ml and 1.3-5.4 micrograms/ml, respectively) than maternal concentrations, with fetal-to-maternal plasma ratios of 0.63-1.0 for AZT. Fetal tissue distribution of tritium was highest in the kidney and lowest in the brain. Although the active triphosphorylated metabolite was not detected in the fetus, the AZT-monophosphate was detected in almost all fetal tissues examined. Our data indicate that AZT is rapidly converted to the glucuronide and monophosphate metabolites in the fetus after maternal infusion.
Animals↗
Kappa opioid receptor-like immunoreactivity in guinea pig brain: ultrastructural localization in presynaptic terminals in hippocampal formation.
Physiological and pharmacological studies have suggested that kappa opioid receptors (KORs) may be located presynaptically in the guinea pig hippocampal formation. In the present study, KOR-like immunoreactivity (-LI) was examined by using a rabbit antibody raised against a synthetic peptide from the carboxyl terminus of a cloned rat kappa receptor (KT). The specificity of affinity-purified KT antibody was confirmed by Western blotting, enzyme-linked immunosorbent assay, immunolabeling of KORs expressed in Xenopus oocytes, and immunocytochemical preadsorption controls. Specificity also was demonstrated by the light microscopic distribution of KT-LI in sections through the forebrain and the pons, which was largely consistent with the distribution of KORs previously reported, and resembled that of immunoreactivity for dynorphin B, an endogenous ligand for KORs. Detailed analysis of the hippocampal formation revealed that KT-LI was located predominantly in thin processes in the granule cell and inner molecular layers of the dentate gyrus. A few KT-labeled processes were also present in stratum lacunosum-moleculare of the CA1 region and all layers of the CA3 region of the hippocampus. By electron microscopy, KT-LI was restricted to unmyelinated axons and axon terminals, and was associated with plasma membranes, large dense-core vesicles, and cytoplasmic surfaces of small vesicles. In the dentate gyrus, immunolabeled terminals formed asymmetric synapses with granule cell perikarya and large unlabeled dendrites. In the CA3 region of hippocampus, KT-LI was present in small unmyelinated axons. The results of this study 1) demonstrate the specificity of the KT antibody, 2) show that the distribution of KT labeling corresponds well with previous KOR and dynorphin localization in many regions, and 3) provide ultrastructural evidence that KORs are located presynaptically in the guinea pig hippocampal formation.
Animals↗
The effects of taurine in a rodent model of aging.
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Aging↗
Recombinant human dihydroorotate dehydrogenase: expression, purification, and characterization of a catalytically functional truncated enzyme.
An N-terminally truncated cDNA for human dihydroorotate dehydrogenase (DHODase) was placed under the control of the inducible T7 lac promoter in a pyrimidine auxotrophic strain of Escherichia coli lacking the endogenous enzyme. Induction of gene expression rescued growth in media lacking exogenous pyrimidines. The recombinant enzyme was purified to homogeneity from detergent extracts of bacterial membranes by two chromatographic steps. The purity of the resulting enzyme was judged to be > 95% based on SDS-PAGE with Coomassie staining. The enzyme displays an apparent molecular weight of ca. 40 kDa on SDS-PAGE and ca. 120 kDa on native size-exclusion chromatography, suggesting that the native enzyme is multimeric. Recombinant DHODase displayed a specific activity and Km for dihydroorotate that were similar to those for the enzymes from bovine and human liver tissue. The pH dependence of the activity of the recombinant enzyme was likewise similar to that of the enzyme from human liver and may indicate the involvement of a critical histidine residue in catalytic turnover; only eight histidine residues remain in the truncated version of DHODase used here. The catalytic activity of the recombinant enzyme is inhibited in a dose-dependent fashion by the histidine-selective modifying agent diethylpyrocarbonate. These results further suggest a potential role for histidine in enzyme turnover. Brequinar sodium, an experimental drug which has been shown to be a nanomolar noncompetitive inhibitor of mammalian DHODases, inhibited the activity of the purified recombinant enzyme with a Ki value similar to that for enzyme derived from human liver tissue. The recombinant DHODase thus displays enzymatic behavior similar to the 50-kDa full-length human liver enzyme, illustrating that the catalytically essential structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enzyme that was expressed here.
Amino Acid Sequence↗
Endogenous excitatory amino acid release from brain slices and astrocyte cultures evoked by trimethyltin and other neurotoxic agents.
Trimethyltin (TMT) is a toxic alkyltin compound that is known to produce neuronal necrosis in the CNS. The present study examined the effects of TMT on the release of excitatory amino acids (EAA) from cortical slices prepared from adult and aged (24 months old) rats. The calcium dependence of TMT-induced EAA efflux was evaluated and compared to other neurotoxic agents. The actions of TMT were also evaluated in an astrocyte culture model to assess glial contributions to TMT-induced EAA efflux. TMT (10-1000 microM) evoked a dose-related increase in GLU and ASP efflux during a 30 min incubation period and this efflux was sustained or slightly higher during a 15 min recovery period. TMT-stimulated GLU efflux was not altered in aged rats. TMT-induced GLU efflux was significantly reduced by removing extracellular calcium and including 10 microM EGTA in the incubation media. Calcium channel blockers (nifedipine, verapamil, flunarizine, amiloride, neomycin) and MK-801 did not significantly attenuate TMT-induced GLU efflux. Diltiazem (25 microM) produced modest but inconsistent reductions in TMT-induced GLU efflux from brain slices, and significantly inhibited the leakage of lactate dehydrogenase (LDH) from TMT-treated astrocyte cultures. TMT did not increase GLU efflux from glial cultures during a 30 min incubation period, but did significantly elevate GLU efflux during the 15 min recovery period. TMT evoked the release of EAA by both calcium dependent and independent mechanisms in brain slices. TMT at high concentrations also produced a delayed increase in glial GLU efflux. These studies suggest that excitotoxic mechanisms may contribute to TMT-induced neurotoxicity.
Aging↗