Extrathyroidal conversion of thyroxine to 3,3',5'-triiodothyronine (reverse-T3) and to 3,5,3'-triiodothyronine (T3) in humans

In order to estimate the relative magnitude of the two alternative pathways of monodeiodination of thyroxine (T4) in adult humans, the metabolic clearance rates (MCR) and production rates (PR) of 3,3',5'-triiodothyronine (reverse-T3,rT3) and of 3,5,3'-triiodothyronine (T3) were determined in six euthyroid control subjects (C) and in five hypothyroid patients (H) receiving L-T4 as replacement therapy (0.15-0.3 mg/day). MCR was computed by a non-compartmental method of analysis from the plasma disappearance of 125I rT3 and 131I T3 during 72 h following simultaneous injection of tracers. PR was calculated from MCR and the serum concentration of rT3 and T3, respectively, determined by radioimmunoassay. In the H subjects, rT3 MCR averaged 97.1 +/- 12.8 (SD) 1/day and rT3 PR, 34.3 +/- 12.8 microng/day; T3 MCR was 28.7 +/- 6.1 1/day and T3 PR, 20.3 +/- 6.6 microng/day (all corrected to 70 kg body weight). These results were not significantly different from those in the control group; rT3 MCR 104 +/- 24 1/day, rT3 PR 33.0 +/- 9.2 microng/day; T3 MCR 24.0 +/- 5.9, T3 PR 24.2 +/- 4.1. The proportionof total triiodothyronine (rT3 averaged 62% in H patients and was similar (57%) in the C group. The results obtained in the H subjects indicate that the production of rT3 is a major route of T4 metabolism, equal to or exceeding that of T3. From the close agreement between the mean values for rT3 PR in the C and H groups it is concluded that most, if not all of the rT3 produced in normal humans is derived by extrathyroidal conversion from T4.     

Radioimmunoassay for 3,3',5'-triiodothyronine (reverse T3, R-T3) in unextracted human serum

Highly specific antibodies against 3,3',5'-triiodothyronine (reverse T3, R-T3) have been produced in rabbits. The crossreaction with T4 is about 0.05%. A radioimmunoassay for R-T3 in unextracted serum was developed. ANS is used for blocking the binding of tracer and endogenous R-T3 to TBG. The sensitivity to the assay is 0.06 ng/ml plasma. The mean normal R-T3 concentration is 0.20 ng/ml. Thyrotoxic patients show elevated levels; in most hypothyroid patients R-T3 concentrations are below the detection limit.     

The effect of (2'-5') and (3'-5') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.     

p59OASL, a 2'-5' oligoadenylate synthetase like protein: a novel human gene related to the 2'-5' oligoadenylate synthetase family

The 2'-5' oligoadenylate synthetases form a well conserved family of interferon induced proteins, presumably present throughout the mammalian class. Using the Expressed Sequence Tag databases, we have identified a novel member of this family. This protein, which we named p59 2'-5' oligoadenylate synthetase-like protein (p59OASL), shares a highly conserved N-terminal domain with the known forms of 2'-5' oligoadenylate synthetases, but differs completely in its C-terminal part. The C-terminus of p59OASL is formed of two domains of ubiquitin-like sequences. Here we present the characterisation of a full-length cDNA clone, the genomic sequence and the expression pattern of this gene. We have addressed the evolution of the 2'-5' oligoadenylate synthetase gene family, in the light of both this new member and new 2'-5' oligoadenylate synthetase sequence data from other species, which have recently appeared in the databases.     

Effects of 3,3',4,4'-tetrachlorobiphenyl, 2,3,3',4,4'-pentachlorobiphenyl, and 3,3',4,4',5-pentachlorobiphenyl on the developing chicken embryo when injected prior to incubation

Great Lakes waterbird populations have experienced less-than-expected hatchability of eggs and a greater-than-expected incidence of developmental abnormalities. Such deleterious effects have been attributed to polyhalogenated hydrocarbons such as polychlorinated biphenyls (PCBs). PCBs are of primary concern since they are present in significant quantities in the environment. Specific PCB congeners, 3,3',4,4',5-pentachlorobiphenyl (IUPAC number 126), 3,3',4,4'-tetrachlorobiphenyl (IUPAC number 77), and 2,3,3',4,4'-pentachlorobiphenyl (IUPAC number 105), were injected (singly or in combination) into the yolks of White Leghorn chicken (Gallus domesticus) eggs prior to incubation. Teratogenicity was assessed in dead embryos and in hatchlings. Hatchlings were raised for 3 wk to assess body weight gain and mortality. At the end of the 3-wk period, chicks were subjected to necropsy and the brain, bursa, heart, liver, spleen, and testes were removed and weighed. All 3 congeners caused increased embryo mortality, with approximately 50% mortality occurring at 0.6, 8.8, and 5592 micrograms/kg egg for congeners 126, 77, and 105, respectively. All three congeners also produced significantly more abnormalities than the vehicle. Chicks from PCB-injected eggs had lower body weights at wk 2 and 3 of age. Congener 126 caused lower relative bursa weights, congener 77 caused greater relative spleen weights and lower relative liver weights, and all three congeners caused relative heart weights to be greater when compared to control.     

Effects of the antiestrogenic environmental pollutant 3,3',4,4', 5-pentachlorobiphenyl (PCB #126) in rat bone and uterus: diverging effects in ovariectomized and intact animals

The purpose of this study was to compare effects on rat bone and uterus of estrogen depletion and exposure to the coplanar PCB-congener 3,3',4,4',5-pentachlorobiphenyl (PCB #126) which exhibits anti-estrogenic properties. Half of the rats were ovariectomized (n = 20) and the other half were sham-operated. Ten of the ovariectomized rats and ten of the sham operated were exposed to PCB #126 (ip injections) for 3 months (total dose: 384 microgram/kg body wt). The remaining control rats were injected with corn oil (vehicle). The rats were killed and the tibiae and uteri were dissected. The left tibia was used for measurements of weight, length, and bone mineral density and the right for histomorphometrical analysis. The uteri were analyzed with respect to estrogen receptor content. PCB #126 exposure did not affect bone mineral density or trabecular bone volume of tibia in sham-operated rats. In ovariectomized rats PCB #126 exposure resulted in a decreased length and an increased bone mineral density of tibia. An obvious PCB #126 induced increase in osteoid surface was observed in sham-operated rats. The cortical thickness and the organic content of the tibia were also increased in these rats. In estrogen deprived tissue like the uteri of ovariectomized rats, PCB #126 showed weak estrogen agonistic activity. The observed effects of PCB #126 on bone and uterine tissues differed between ovariectomized and sham-operated rats.     

Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (PCB153) in guinea pig

1. The in vitro and in vivo metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (PCB153) in guinea pig has been studied. 2. Seven metabolites were detected in the faeces of PCB153-treated animals and three were identical to those produced by dog liver microsomes. The detection of a metabolite where a chlorine atom was shifted from the 2- to 3-position strongly suggested the involvement of 2,3-arene oxide intermediate, and evidence for the concomitant formation of a 3,4-arene oxide intermediate was provided by identifying other two minor metabolites which were dechlorinated at the 4-position. 3. In vitro studies using liver microsomes from guinea pigs revealed that the 2,3-arene oxide and 3-hydroxylation pathways are the predominant metabolic routes compared with the 3,4-arene oxide pathway. Although the guinea pig is an another species that can metabolize PCB153 mainly to the 2,3-arene oxide intermediate, the rate of formation was only about one-tenth of the dog. 4. These results indicate that the ability to form this unusual 2,3-arene oxide intermediate may not be responsible for high excretion rate of this congener. Our data also suggest that the cytochrome P450-catalysed metabolism of PCB153 in the guinea pig and dog are similar, whereas for post-cytochrome P450 metabolism, the guinea pig resembles the rabbit.     

Platelet aggregation inhibiting and anticoagulant effects of oligoamines, XX: 4,4',4'-(1,3,5)-benzene-tris-sydnone imines

The synthesis of ten tris-sydnone imine derivatives, unknown up to now, is described. All compounds are alkyl or arylalkyl substituted in 3-position of the sydnone imine. The most powerful agent was the 3-propyl derivative 6c. It inhibits the aggregation of human platelets induced by collagen in a concentration of 1 mumol/L half maximally. Its N-ethoxycarbonyl derivative 7c, which was designed as a prodrug, showed only small antithrombotic effects in rats. The reason for this low degree of activity is discussed.     

Orf186 represents a new member of the Nudix hydrolases, active on adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH

orf186, a new member of the Nudix hydrolase family of genes, has been cloned and expressed, and the protein has been purified and identified as an enzyme highly specific for compounds of ADP. Its three major substrates are adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH, all implicated in a variety of cellular regulatory processes, supporting the notion that the function of the Nudix hydrolases is to monitor the concentrations of reactive nucleoside diphosphate derivatives and to help modulate their accumulation during cellular metabolism.     

Short-term effects of thyroid hormones on the Na/H antiport in L-6 myoblasts: high molecular specificity for 3,3',5-triiodo-L-thyronine

The thyroid hormones L-T3 and L-T4 were shown to activate the Na/H antiport in L-6 cells from rat skeletal muscle by a rapid, nongenomic mechanism. Under pH equilibrium conditions, a significant rise in the intracellular pH, measured by the fluorescent pH indicator 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein was observed after the addition of physiological concentrations (10(-10) M) of either L-T3 or L-T4, but with different time courses. L-T3 at all concentrations increased the pH after a delay of 2 min, whereas L-T4 showed a concentration-dependent lag time, going from 11 min at 10(-11) M down to 5 min for a hormone concentration of 10(-6) M. The effect of L-T4 was blocked in the presence of the 5'-deiodinase inhibitor 6-n-propyl-2-thiouracil, suggesting that the difference in lag time between L-T3 and L-T4 was due to the 5'-deiodination process that transforms L-T4 into the bioactive L-T3. In short term studies (<5 min), a high molecular specificity for L-T3 was found, as L-T4, rT3, the D-isomer of T3, and the deaminated analogues were ineffective at physiological concentrations. In analogy with the results found at equilibrium, intracellular pH recovery from an acid load and set-point were increased after 2 min for L-T3 (10(-9) M) and after 10 min for L-T4 (10(-9) M). The effect of the hormones on the intracellular pH was completely blocked by the specific antiport inhibitor 5-(ethyl-N-isopropyl)amiloride. These findings suggest that thyroid hormones may play an active role in the recovery from muscular acidosis through direct stimulation of the Na/H antiport.     

Reduced variation of tracer binding in digoxin radioimmunoassay by use of (125I)-labeled tyrosine-methyl-ester derivative: relation of thyroxine concentration to binding

Between-sample variation in tracer binding in the 125I-labeled digoxin radioimmunoassay was investigated with two tracers, 3-O-succinyl-digoxigenin-[125I]-labeled tyrosine and [125I]-labeled tyrosine-methyl-ester-digoxin. Digoxin-free serum samples having various concentrations of thyroxine were assayed with both tracers. The percentage of tracer bound when the samples were assayed with the first-mentioned tracer was increased significantly for the low thyroxine groups when compared to the normal (P less than 0.001) or the high thyroxine groups (P less than 0.05). Little difference existed when the latter tracer was used. There was variation in tracer binding when serum from dogs dosed with thyrotropin was assayed with the first tracer, but there was little variation with the second. Tracer binding may be influenced by thyroxine-binding proteins. Variation in tracer binding appears to be reduced when [125I]-labeled tyrosine-methyl-ester-digoxin is used.     

phi29 DNA polymerase residue Ser122, a single-stranded DNA ligand for 3'-5' exonucleolysis, is required to interact with the terminal protein

Three amino acid residues highly conserved in most proofreading DNA polymerases, a phenylalanine contained in the Exo II motif and a serine and a leucine belonging to the S/TLx2h motif, were recently shown to be critical for 3'-5' exonucleolysis by acting as single-stranded DNA ligands (de Vega, M., Lázaro, J.M., Salas, M. and Blanco, L. (1998) J. Mol. Biol. 279, 807-822). In this paper, site-directed mutants at these three residues were used to analyze their functional importance for the synthetic activities of phi29 DNA polymerase, an enzyme able to start linear phi29 DNA replication using a terminal protein (TP) as primer. Mutations introduced at Phe65, Ser122, and Leu123 residues of phi29 DNA polymerase severely affected the replication capacity of the enzyme. Three mutants, F65S, S122T, and S122N, were strongly affected in their capacity to interact with a DNA primer/template structure, suggesting a dual role during both polymerization and proofreading. Interestingly, mutant S122N was not able to maintain a stable interaction with the TP primer, thus impeding the firsts steps (initiation and transition) of phi29 DNA replication. The involvement of Ser122 in the consecutive binding of TP and DNA is compatible with the finding that the TP/DNA polymerase heterodimer was not able to use a DNA primer/template structure. Assuming a structural conservation among the eukaryotic-type DNA polymerases, a model for the interactions of phi29 DNA polymerase with both TP and DNA primers is presented.     

Receptor cross-talk can optimize assays for autoantibodies to the thyrotropin receptor: effect of phenylisopropyladenosine on adenosine 3',5'-monophosphate and inositol phosphate levels in rat FRTL-5 thyroid cells

Immunoglobulins (IgG) from patients with Graves' disease increase inositol phosphate (IP) as well as cAMP production in rat thyroid FRTL-5 cells; IgGs from normal control subjects do not. Graves' IgG-and TSH-induced IP formation is inhibited by blocking TSH receptor (TSHR) antibodies from hypothyroid patients with primary myxedema, as is the cAMP response; this suggests that the Graves' IgG are acting through the TSHR to induce both the cAMP and phosphatidyl-inositol 4,5-biphosphate signal cascades in FRTL-5 thyroid cells as in cells with recombinant TSHR. Optimal conditions for measuring the Graves' IgG-induced IP increase include a NaCl-free Hanks' Balanced Salt Solution (HBSS) buffer system and a P1 purinergic receptor agonist; the action of each is additive. Optimization by NaCl-free HBSS is similar to that observed in cAMP assays and is specific for TSH or Graves' IgG; thus, NaCl-free HBSS did not affect ATP-induced, and actually inhibited norepinephrine-induced, IP production in FRTL-5 cells. The P1 purinergic receptor agonist acts via receptor cross-talk, which also allows further optimization of cAMP assays. Thus, adenosine deaminase improves Graves' IgG-induced cAMP production by removing adenosine from the medium. Although NaCl-free HBSS improved TSH- or Graves' IgG-induced IP and cAMP production in cells with recombinant TSHR; the modulatory action of phenylisopropyladenosine was lost.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Disposition of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) in rats

The disposition of diadenosine 5'5"'-P1,P4-tetraphosphate (Ap4A), an endogenous dinucleotide, was investigated in rats. The degradation of Ap4A in rat plasma was very rapid and could be explained by a Michaelis-Menten equation: Km and Vmax values were 1.69 micrograms/ml and 4.32 micrograms/min/ml, respectively. Ap4A was degraded in rat plasma to ATP and AMP, but not to 2 ADP molecules, and these nucleotides were further degraded through adenosine. The degradations kinetics were examined. After intravenous bolus injection, Ap4A in plasma declined rapidly and the rate of elimination was dose-dependent: the biological half-life was about 3s at the dose of 1 mg/kg and was longer at 3 mg/kg. When Ap4A was administered by intravenous infusion (0.5 or 1.0 mg/kg/min), the plasma level rapidly reached a steady-state, which then rapidly declined after stopping the infusion.     

Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.     

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(beta, gamma-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.