Effects of streptozotocin-induced diabetes mellitus on hypothalamic-pituitary-thyroid axis in rats

The effects of streptozotocin-induced diabetes mellitus on the hypothalamic-pituitary-thyroid axis in rats were studied. Streptozotocin (60 mg/kg) was injected ip. Rats were decapitated at two and four weeks after the streptozotocin treatment. Thyrotropin releasing hormone (TRH), thyrotropin (TSH), thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2) and 3',5'-diiodothyronine (3',5'-T2) were measured by means of the specific radioimmunoassay for each. Immunoreactive TRH (ir-TRH) contents in the hypothalamus significantly decreased at four weeks (p less than 0.02). Basal TSH levels in plasma significantly decreased (p less than 0.005, p less than 0.001), and plasma ir-TRH and TSH responses to cold were significantly inhibited after the streptozotocin treatment (p less than 0.001). The plasma TSH response to TRH was decreased, but not significantly. The plasma T4 and T3 levels fell significantly. RT3 did not change throughout the experiment. 3,3'-T2 levels in plasma fell significantly, whereas 3',5'-T2 increased. Blood glucose levels rose significantly after streptozotocin treatment, but insulin treatment led to partial restoration. The findings suggest that streptozotocin-induced diabetes mellitus affects various sites of the hypothalamic-pituitary-thyroid axis in rats.     

Triiodothyronamine--a beta-adrenergic metabolite of triiodothyronine?

Triiodothyronamine (Triam) is a potential metabolite of triidothyronine (T3), resulting from decarboxylation of the side-chain. In an attempt to elucidate the physiological properties of Triam we have investigated the binding of Triam to beta-adrenergic receptors, using turkey-erythrocytes and performing binding studies with ( (-)(3H)-dihydroalprenolol) ( (-)(3H)-DHA) as a specific beta-adrenergic ligand. The inhibition constant Ki for Triam was determined as 5 X 10(-6) M, compared to dopamine (Ki = 1,3 X 10(-2) M), norepinephrine (Ki = 3 X 10(-4) M), epinephrine (Ki = 5 X 10(-5) M) and isoproterenol (Ki = 3 X 10(-6) M). The inhibition of ( (-)(3H)-DHA)-binding by Triam was further compared with other iodothyronines thyroxine (T4), T3, 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (3,3'-T2). It is concluded that Triam binds to beta-adrenergic receptors like naturally occurring amines but different from typical circulating iodothyronines.     

Effect of ethanol and linoleic acid on changes in biliary excretion of iodothyronines possibly related to thyroxine deiodination in rat liver

Groups of male rats weighing about 350 g were inserted polyethylene tubings into bile duct and femoral vein under pentobarbital anesthesia. Several iodothyronines (i.e. T4, T3, rT3, 3,5-T2, 3,3'-T2 and 3',5'-T2) were estimated in 2-hr portions of bile with the aid of specific radioimmunoassay. After the infusion of ethanol (0.3 ml/hr/rat for 4 hr) an increase of biliary excretion of rT3 and a decrease of 3,5-T2 was found as compared to controls. When 5 mg linoleic acid was added to 1.2 ml ethanol, the increase of rT3 was significantly higher than that after ethanol only and, in addition, significant increase of 3',5'-T2 excretion was found. It was concluded that both ethanol and unsaturated fatty acids may inhibit 5'-monodeiodination in the liver and that unsaturated nonesterified fatty acids may exert such effect even when administered intravenously without underlying metabolic disorders.     

Effects of propylthiouracil and methylmercaptoimidazol on metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells.

Monkey hepatocarcinoma cell monolayer cultures (NCLP-6E) metabolized thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine by phenolic and nonphenolic ring deiodinations and sulfation of the deiodinated products, as shown in previous work with this system. The effects of the antithyroid drugs, propylthiouracil (PTU) and methylmercaptoimidazole (MMI), on these processes was investigated. PTU, at 0.1 and 1 mM, inhibited only phenolic ring deiodination. MMI at 1 mM had no effect, but 32 mM inhibited deiodination of both rings as well as sulfation. The findings suggest that the increased serum rT3 level caused by PTU in vivo is the result of decreased rT3 deiodination, in contrast to the increased rT3 production which is caused by starvation.     

Serum concentrations of 3, 3'-diiodothyronine, 3', 5'-diiodothyronine, and 3, 5-diiodothyronine in altered thyroid states

To investigate the thyroid hormone metabolism in altered states of thyroid function, serum concentrations of 3, 3'-diiodothyronine (3, 3'-T2), 3', 5'-T2 and 3, 5-T2 as well as T4, T3 and rT3 were determined by specific radioimmunoassays in 17 hyperthyroid and 10 hypothyroid patients, before and during the treatment. Serum T4, T3, rT3, 3, 3'-T2 and 3', 5'-T2 concentrations were all higher in the hyperthyroid patients than in age-matched controls and decreased to the normal ranges within 3 to 4 months following treatment with antithyroid drugs. In the hypothyroid patients, these iodothyronine concentrations were lower than in age-matched controls and returned to the normal ranges after 2 to 3 months treatment with T4. In contrast, serum 3, 5-T2 concentrations in hyperthyroid patients (mean +/- SE : 4.0 +/- 0.5 ng/dl) were not significantly different from those in controls (3.9 +/ 0.4 ng/dl), although they tended to decrease in 3 of 6 patients after the antithyroid drug therapy. Serum 3, 5-T2 levels in the hypothyroid patients (3.8 +/- 0.6 ng/dl) were also within the normal range and showed no significant change following the T4 replacement therapy. However, serum 3, 5-T2 as well as 3, 3'T2 concentrations rose significantly with a marked rise in serum T3 following T3 administration, 75 micrograms/day for 7 days, in Graves' patients in euthyroid state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroxine inactivation by starvation in cultured hepatocarcinoma cells, Formation of reverse triiodothyronine.

The effect of starvation on thyroid hormone metabolism was studied in monkey hepatocarcinoma monolayer cultures. Nonphenolic ring monodeiodination of thyroxine, 3, 5, 3'-triiodothyronine and 3, 3'-diiodothyronine was accelerated. Since phenolic ring deiodination of 3, 3',5'-triiodothyronine (reverse T3) was unaffected, this metabolite accumulated in the medium during thyroxine metabolism. This suggests that increased serum reverse T3 in malnourished humans may be caused by enhanced deiodination of thyroxine rather than decreased rT3 catabolism.     

Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.     

Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases.

Sulfation is an important pathway in the metabolism of thyroid hormones. Sulfated iodothyronines are elevated in nonthyroidal illnesses and in the normal human fetal circulation. We assayed and characterized COS-1 cell expressed recombinant human liver dehydroepiandrosterone sulfotransferase (DHEA ST or SULT2A1) and estrogen sulfotransferase (EST or SULT1E1) activities for the first time with triiodothyronine (T(3)) as the substrate. Several biochemical properties that included apparent K(m) values, thermal stabilities, and responses to the inhibitors 2, 6-dichloro-4-nitrophenol and NaCl were tested. SULT2A1, a member of the hydroxysteroid sulfotransferase family, used 3,3'-T(2) more readily than T(3) and 3,5-T(2) as substrates, but had the lowest apparent K(m) value for T(3) of any reported human SULT. SULT1E1, a member of the phenol sulfotransferase family, used 3,3'-T(2) and rT(3) more readily than T(3), and also displayed the greatest specificity for T(4) among human SULTs. SULT2A1 may contribute more to iodothyronine sulfation than previously suspected. Potential roles of both steroid sulfotransferases in the enhanced sulfation of nonthyroidal illnesses and fetal development invite further investigation.     

Purification and characterization of N-acetylglucosaminyl sulfotransferase from chick corneas

Sulfation is an important conjugation pathway in deactivating thyroid hormones, keeping the proper hormonal balance, and increasing the rate of thyroid hormone metabolism. We have identified, cloned, and characterized a sulfotransferase (SULT) that is capable of thyroid hormone conjugation in the dog. This enzyme, designated cSULT1B1, displays a strong identity (>84%) to the human ST1B2 enzyme. However, cSULT1B1 displays less identity, about 73%, to mouse and rat orthologs. In addition, the canine enzyme is three amino acids shorter than the rodent ones but has the same length as the human ortholog, 296 amino acids. The bacterial expressed and partial purified cSULT1B1 enzyme sulfates p-nitrophenol and 1-naphtol, but not dopamine. The thyroid hormones 3,3'-diiodothyronine and 3,5,3'-triiodothyronine are efficiently sulfated. 3,3',5'-Triiodothyronine is sulfated to lesser degree while sulfation of 3,5'-diiodothyronine and 3,3',5,5'-tetraiodothyronine cannot be detected. The cSULT1B1 is found in the colon (highest level), kidney and small intestine in dogs, but surprisingly not in the male dog liver although low levels of immunoreactivity were detected in the female dog liver. The male dog expresses more of SULT1B1 enzyme in the lower part of the small intestine while the female dog displays an opposite pattern of expression. These results describe the cloning and characterization of a canine thyroid hormone sulfating enzyme that is more closely related to the human ortholog than to the rodent thyroid sulfating enzymes.     

NMR studies of echinomycin bisintercalation complexes with d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution: sequence-dependent formation of Hoogsteen A1.T4 and Watson--Crick T1.A4 base pairs flanking the bisintercalation site

We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-dependency of iodothyronine metabolism in isolated perfused rat liver.

1. Isolated livers from fed male rats were perfused for 2 h with T4 (L-thyroxine), T3 (L-3,3',5-tri-iodothyronine) or rT3 (L-3,3',5'-tri-iodothyronine) at different pH values (7.1--7.6) in a fully synthetic medium, whereby normal metabolic functions were maintained without addition of rat blood constituents or albumin. 2. T3 output into the medium and net T3 production reached a maximum at a pH of the medium of 7.2 and significantly decreased with alteration of the pH when livers were perfused with T4 as a substrate. 3. However, the net T4 and T3 uptake by the liver, as well as the hepatic T4 and T3 content after perfusion, were not dependent on the pH of the perfusion when livers were offered T4 or T3 as substrates respectively. 4. Determination of intracellular pH by the analysis of the distribution of the weak acid dimethyloxazolidinedione allows the conclusion that the pH optimum of iodothyronine 5'-deiodinase in the intact perfused liver corresponds to the maximum determined in vitro for the membrane-bound enzyme localized in the endoplasmic reticulum. 5. The rapid 5'-deiodination of rT3 to 3,3'-T2 (L-3,3'-di-iodothyronine), the fast disappearance of 3,3'-T2, and the fact that no net rT3 production from T4 could be detected, supports the hypothesis that in rat liver iodothyronine 5'-deiodinase activity seems to predominate over iodothyronine 5-deiodinase activity. 6. Thus the rat liver can be considered in normal physiological situations as an organ forming T3 from T4 and deiodinating rT3 originating from extrahepatic tissues, whereby the cellular iodothyronine 5'-deiodination rate is controlled by the intracellular pH.     

Existence of extrathyroidal conversion inhibitor (IEC) in starved animals and its influence on thyroid hormones deiodination in liver and kidney in vitro

To find out whether an inhibitor of extrathyroidal conversion of iodothyronines is present in sera of starved animals, pig liver and kidney homogenates were incubated with T4, T3 or rT3 and dithiotreitol in the presence of evaporated diethyl ether extracts of sera obtained from fed and starved (1-12 days) rabbits. Sera extracts of short-term (1-4 days) starved rabbits caused a significant inhibition of T4 to T3 conversion (54% on day 3) and T4 to rT3 deiodination (52% on day 2) in liver homogenates. Extracts of sera from long-term (8 and 12 days) starved animals diminished only liver T4 to T3 conversion on day 8 and had no influence on liver T4 to rT3 conversion. 5'-deiodination of rT3 (to 3,3'-T2) in liver was gradually decreased by extracts of sera from animals starved during 2-12 days. Liver rT3-5-deiodination (to 3',5'-T2) was significantly impaired on day 4 and totally depressed by long-term starvation. In vitro T3 to 3,3'-T2 conversion in liver was markedly (59-103%) increased by ether extracts of sera from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term fasted rabbits and considerably inhibited (62-72%) by long-term fasting. T4 to T3 conversion in kidney was significantly influenced by sera extracts obtained neither from short-term nor from long-term fasted rabbits but T4-5-deiodination (to rT3) was reduced by sera extracts of short-term fasted animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism

Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T(3) levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T(3) and T(4) but little or no increase in rT(3) or 3,3'-diiodothyronine (3,3'-T(2)) uptake. MCT8 expression produced large increases in T(4) metabolism by cotransfected D2 or D3, T(3) metabolism by D3, rT(3) metabolism by D1 or D2, and 3,3'-T(2) metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[(125)I]T(3). hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T(3). Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability.     

Multinuclear nuclear magnetic resonance studies of Na cation-stabilized complex formed by d(G-G-T-T-T-T-C-G-G) in solution. Implications for G-tetrad structures.

There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment and report on multinuclear 1H, 31P and 13C nuclear magnetic resonance studies on the d(G2T4CG2) deoxynonanucleotide with Na cation as counterion in aqueous solution at low temperature. This sequence forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation (see the paper on d(G2T5G2) describing optical and calorimetric measurements by Jin, R., Breslauer, K. J., Jones, R. A. & Gaffney, B. L. (1990), Science, 250, 543-546). The four narrow exchangeable protons detected between 11.5 and 12.0 parts per million (p.p.m.), which are common to the d(G2T4CG2) deoxynonanucleotide and the d(G2TCG2) deoxyhexanucleotide sequences, are assigned to deoxyguanosine imino protons hydrogen-bonded to carbonyl acceptor groups. These narrow imino protons are not detected for d(IGN5IG) and d(I2N5G2), where two deoxyguanosine residues are replaced by two deoxyinosine residues in the deoxynonanucleotide sequences. This implies that the 2-amino protons of deoxyguanosine must also participate in hydrogen bond formation and stabilize the structured conformation of d(G2T4CG2) in Na cation-containing solution. We have completely assigned the base and sugar H1', H2',2', H3', and H4' protons of the d(G2T4CG2) oligomer following analysis of two-dimensional nuclear Overhauser enhancement spectroscopy and two-dimensional correlated spectroscopy data sets in 0.1 M-NaCl, 10 mM-sodium phosphate, 2H2O solution at 0 degree C. The relative magnitude of the nuclear Overhauser enhancements (NOEs) between the base H8 and its own sugar H1' protons of individual deoxyguanosine residues establishes that G1 and G8 adopt syn orientations while G2 and G9 adopt anti orientations about the glycosidic bond in the d(G1-G2-T3-T4-T5-T6-C7-G8-G9) sequence in both Na and K cation-containing aqueous solution. Consequently, any structure proposed for the tetramolecular complex of d(G2T4CG2) must exhibit alternating G(syn) and G(anti) glycosidic torsion angles within each strand. The directionality and magnitude of the observed NOEs are consistent with the G(syn)-G(anti) steps adopting right-handed helical conformations in solution. We also note that the H8 protons of G1 and G8 (7.35 to 7.45 p.p.m.) in a syn alignment are shifted significantly upfield from the H8 protons of G2 and G9 (8.0 to 8.3 p.p.m.) in an anti alignment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex

High-resolution proton and phosphorus NMR studies are reported on the self-complementary d(C1-G2-T3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplex (henceforth called O6meG.T 12-mer), which contains T3.O6meG10 interactions in the interior of the helix. The imino proton of T3 is observed at 9.0 ppm, exhibits a temperature-independent chemical shift in the premelting transition range, and broadens out at the same temperature as the imino proton of the adjacent G2.C11 toward the end of the helix at pH 6.8. We observed inter base pair nuclear Overhauser effects (NOEs) between the base protons at the T3.O6meG10 modification site and the protons of flanking G2.C11 and G4.C9 base pairs, indicative of the stacking of the T3 and O6meG10 bases into the helix. Two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) studies have permitted assignment of the base and sugar H1', H2', and H2' nonexchangeable protons in the O6meG.T 12-mer duplex. The observed NOEs demonstrate an anti conformation about all the glycosidic bonds, and their directionality supports formation of a right-handed helix in solution. The observed NOEs between the T3.O6meG10 interaction and the adjacent G2.C11 and G4.C9 base pairs at the modification site exhibit small departures from patterns for a regular helix in the O6.meG.T 12-mer duplex. The phosphorus resonances exhibit a 0.5 ppm spectral dispersion indicative of an unperturbed phosphodiester backbone for the O6meG.T 12-mer duplex. We propose a model for pairing of T3 and O6meG10 at the modification site in the O6meG.T 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Placental outer and inner ring monodeiodination of thyroxine and triiodothyronines in the rabbit

Both inner- and outer-ring iodothyronines deiodinating activity was found in homogenates of rabbit placentas. The T4 to rT3 and T3 to 3,3'-T2 deiodinating activity was already high on day 10 before delivery but decreased being about 7 times lowered on day 5. Once the T3 to 3,3'-T2 monodeiodination reached a low and a relatively steady level, the outer ring deiodination of T4 begun, reaching a peak value at about day 3 before term and then fell again. The fetal serum thyroid hormones levels were low, showing no significant variability during the period of observation. The results suggested that in the rabbit, representing animals in which the thyroid gland activity begins early in fetal life, there are two distinct phases of the placental monodeiodinating activity. The first is characterized by a high inner-ring deiodinating activity (yielding rT3) and is followed by the second phase with a high outer-ring deiodinating activity (yielding T3) declining just before term.     

The effect of iodothyronines on the conversion of thyroxine into 3,3''-5-tri-iodothyronine in isolated rat renal tubules.

Isolated rat renal tubules prepared by collagenase digestion were used to study the effects of 3,3',5'-tri-iodothyronine ('reverse T3', rT3) and other iodothyronines on the formation of 3,3',5-tri-iodothyronine (T3) from thyroxine (T4). rT3 inhibited the conversion with a dose response over the concentration range 1.5nM-1.5microM. The inhibition was competitive in nature. Both 3,3'-di-iodothyronine and 3',5'-di-iodothyronine also inhibited the production of T3 and T4 in isolated rat renal tubules, but tetraiodothyroacetic acid and 3,5-di-iodothyronine were found to have no effect. These experiments demonstrate in an intact cell system that some naturally occurring iodothyronines have significant effects on T4 deiodination.     

Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N2-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9).(G10-T11-A12-C-13-F14-C15 -A16-T17-G-18) X.F 9-mer duplex. Two-dimensional NMR experiments establish that the X.F 9-mer helix is right-handed with Watson-Crick A.T and G.C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4.C15 and G6.C13 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4.C15 and G6.C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N2-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.     

Structural modeling of the distamycin A-d(CGCGAATTCGCG)2 complex using 2D NMR and molecular mechanics

The structure of the distamycin A-d(CGCGAATTCGCG)2 complex has been determined through a combination of SKEWSKY and NOESY 2D NMR experiments and molecular mechanics calculations. NMR data provided upper bounds on many proton-proton pairs. The advantage of the SKEWSKY/NOESY method is that small groups of strongly coupled spins can be treated accurately as isolated systems. The AMBER molecular mechanics package, modified to include the NMR constraints, was used in energy refinements. Distamycin A fits snugly into the 5'-AATT-3' minor-groove binding site. Structural analysis revealed van der Waals contacts between A5, A6, and A18 C2H and drug H3 protons, potential three-center hydrogen bonding between drug amide protons and adenine N3 and thymine O2 atoms analogous to the spine of hydration in the crystal structure of the free DNA, and stacking of the sugar O1' atoms of A6-C21, T7-T20, and, T8-T19, over drug pyrrole rings 1, 2, and 3, respectively. In addition to hydrophobic effects, hydrogen bonding, and electrostatic interactions proposed by others, it is suggested that stacking interactions between DNA sugar O1' atoms and the three drug pyrrole rings contribute to the stability of the complex.     

Thyroid hormone deiodination in tissues of American plaice, Hippoglossoides platessoides: characterization and short-term responses to polychlorinated biphenyls (PCBs) 77 and 126

We have described the tissue distribution and properties of thyroid hormone (TH) deiodination activities of the marine American plaice, Hippoglossoides platessoides. We then studied the 1- or 4-week responses of the plaice liver and brain deiodination activities and the plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels to an intraperitoneal injection (5-500 ng/g) of the polychlorinated biphenyl (PCB) congeners 77 (3,3'-4,4'-tetrachlorobiphenyl) or 126 (3,3',4,4',5-pentachlorobiphenyl). T4 and 3,3'5'-triiodothyronine (rT3) outer-ring deiodination (ORD) activities were greater in liver than in kidney, gill, heart, brain, intestine or muscle; inner-ring deiodination (IRD) activity occurred in all tissues but was consistently higher in brain. Deiodination characteristics (optimal pH, optimal dithiothreitol concentration, responses to inhibitors and apparent Km values of 0.6-4 nM) fell in the same rage as those of low-Km deiodinases in other teleosts. Deiodination activities were maximal when assayed at 25 degrees C but uniformly low over the natural range of 0-9 degrees C. Neither PCB 77 nor PCB 126 altered brain T4ORD activity or plasma T4 levels (P < 0.05). However, at 1 week post injection hepatic T4ORD activity was increased and plasma T3 levels lowered by PCB 77 (5 and 25 ng/g), while hepatic IRD activity was increased by PCB 126 (50 and 500 ng/g). Neither PCB 77, PCB 126 nor selected hydroxylated. PCBs given in vitro compared with T4 for binding sites on plasma proteins or altered hepatic deiodination activity, indicating no direct action on plasma proteins or deiodinases We conclude that plaice TH deiodination tissue distribution and characteristics resemble those of other teleosts. Deiodination activities are low at natural assay temperatures but at 1 week show some responses to PCBs 77 and 126.