Effect of changes in thyroid state on metabolism of thyroxine by rat placenta

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.     

Variation in karyotypes of Dugesia japonica japonica (Turbellaria) from Ôsaka Prefecture,Central Japan

An analysis of the karyotypes of Dugesia japonica japonica Ichikawa et Kawakatsu, 1964, from 30 localities of seven river systems in Ôsaka Prefecture, Central Japan, revealed a total of 26 karyotypes of which 12 are new-found varieties. More than two karyotypes were found in many localities. The mixoaneuploidic triploid karyotype showed the widest distribution (21 localities), orthoploidic diploid karyotype and orthoploidic mixoploid of diploid and triploid karyotype were next (13 localities, respectively), and other karyotypes (triploidic aneuploid, orthoploidic triploid and mixoploid of triploid & tetraploid) were also found. Some correlation of karyotype with elevation was also detected.     

Prostacyclin stimulation of adenylate cyclase activity in human thyroid membranes

Effect of prostacyclin (PGI2) on adenylate cyclase activity in human thyroid membranes was examined. PGI2 caused a dose- and time-dependent production of cyclic AMP (cAMP) with high potency. When GTP was added in concentrations up to 100 uM, the activation of adenylate cyclase by PGI2 was increased. In the assay medium containing 3 mM ATP, 10 uM GTP and nucleotide regenerating system, the replacement of Mg2+ by increasing concentrations of Mn2+ caused a progressive loss of PGI2 as well as TSH-stimulated adenylate cyclase activities, while high concentrations of Mg2+ (12 or 18 mM) slightly suppressed the activity stimulated by either PGI2 or TSH. Both agents had an additive effect on the stimulation of adenylate cyclase activity in the presence of either 6 mM Mg2+ or 6 mM Mn2+. Gamma-globulin fraction containing non-stimulatory TSH receptor antibody which was prepared from a patient with chronic thyroiditis, suppressed only TSH- but not PGI2-stimulation of the adenylate cyclase activity. These results suggest that PGI2 can stimulate the adenylate cyclase activity in human thyroid tissue, and that PGI2-stimulation may be mediated by the different system from TSH-dependent one.     

Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy

The heart mitochondria isolated from a patient with hypertrophic cardiomyopathy associated with mitochondrial encephalomyopathy were analyzed by immunoblotting using specific antibody against each of the purified mitochondrial energy transducing complexes from beef heart. Subunits of NADH-ubiquinone oxidoreductase (Complex I) were markedly decreased and those of cytochrome c oxidase (Complex IV) were decreased to some extent, but the deficiency of any of these subunits was only partial. On the other hand, the contents of subunits of ubiquinol-cytochrome c oxidoreductase (Complex III) were normal. These results suggest that the decreased levels of some of the Complex I subunits might be the primary cause of disorder in this patient.     

An ubiquinone-binding protein in mitochondrial NADH-ubiquinone reductase (Complex I)

An ubiquinone-binding protein (QP) was purified from mitochondrial NADH-ubiquinone reductase (Complex I). Complex I was separated into 3 fragments: a fraction of hydrophobic proteins, that of soluble iron-sulfur protein (IP) and soluble NADH dehydrogenase of flavoprotein by a procedure involving the resolution with DOC and cholate, followed by ethanol and ammonium acetate fractionations. About 40% of the total ubiquinone was recovered in the IP fragment which consisted of 12 polypeptides. The QP was purified from the IP fragment with a hydrophobic affinity chromatography. SDS-polyacrylamide gel electrophoresis showed that the purified QP corresponded to 14-kDa polypeptide of the IP fragment and was a different protein from the QP (12.4 kDa) in Complex III. The purified QP (14 kDa) contained one mol ubiquinone per mol. The ubiquinone-depleted IP fragment could rebind ubiquinone. These results indicate that an ubiquinone-binding site in Complex I is on the 14-kDa polypeptide of the IP fragment.     

Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin

Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.     

Detection and characterization of a novel factor that stimulates DNA polymerase alpha

A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition.     

Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.     

Simulation analysis of excitation conduction in the heart: Propagation of excitation in different tissues

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline (o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containing NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and alpha-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.