Tryptophan Synthetic Pathway and Its Regulation in Chromobacterium violaceum

Extracts of Chromobacterium violaceum catalyzed all of the reactions involved in synthesizing tryptophan from chorismic acid. Tryptophan auxotrophs which had lost any of these activities did not produce the characteristic purple pigment, violacein, when grown on a medium in which tryptophan was limiting. Gel filtration of extracts allowed us to estimate molecular weights for the tryptophan enzymes. All of the enzymes appeared to have molecular weights below 100,000. No enzymes were observed to occur in aggregates. The specific activities of the enzymes of the tryptophan pathway did not change when mutants were grown under conditions of limiting or excess tryptophan. The first enzyme in the pathway, anthranilate synthetase, was subject to feedback control by the end product, tryptophan. Tryptophan acted as a noncompetitive inhibitor with respect to glutamine, one of the substrates for anthranilate synthetase, and as a competitive inhibitor of the reaction when chorismate, the other substrate, was varied. The nonlinearity observed in the Lineweaver-Burk plot in the latter case suggests that there may be more than one chorismate-binding site on anthranilate synthetase.     

Metabolic Changes in Nicotinamide Adenine Dinucleotide in Response to Anthrax Toxin

Bacillus anthracis produces a toxin both in vitro and in vivo which, when injected intravenously into rats, brings about the death of the animals accompanied by gross pulmonary edema. Lung tissue removed prior to death showed, in vitro, a 30% reduction in overall oxidative metabolism (Q(o2)), whereas the nicotinamide adenine dinucleotide (NAD)-independent succinic dehydrogenase remained unaffected. The NAD concentration in the lungs of injected animals was reduced by 50%. Upon addition of NAD, the Q(o2) of lung tissue from injected animals rose to control values. At 45 min after toxin injection, the serum lactate concentration began to rise, showing about a 3.5-fold increase over controls after 75 min. No changes occurred in the pyruvate concentration. These changes may be explained by increased use of the pyruvate for glycolytic energy production with further loss of NAD. Additional experiments with liver, spleen, kidney, and brain tissues showed that the toxin-induced reduction of Q(o2) is an effect specific for lung tissue. Brain tissue showed a significant increase in oxidative metabolism upon the addition of the toxin, whereas the other tissues remained unaffected. It is suggested that a principal effect of the toxin is to inhibit, in lung tissue, the regeneration of NAD in the respiratory chain.     

Experimentelle Untersuchungen über die Beziehung zwischen Schilddrüse und Hypophyse während der Larvalentwicklung und Metamorphose von Xenopus laevis Daudin

Zusammenfassung Die Normalentwicklung von Schilddrüse und Hypophyse während der Larvalentwicklung und Metamorphose von Xenopns laevis Daudin und die Veränderungen des Hypophysenvorderlappens (HVL) und der Schilddrüse auf Behandlung mit Dinitrophenol, Natriumthiocyanat, Methylthiouracil, Kaliumperchlorat und Thyroxin im Überschuß wurden untersucht.Die Selbstdifferenzierungsfähigkeit der Schilddrüse und die Wirkung exogenen Thyroxins auf die Schilddrüse wurde an hypophysektomierten Tieren herausgestellt.Schilddrüse und -Zellen des Hypophysenvorderlappens werden mit Beginn des Hinterbeinwachstums funktionsfähig. Zur gleichen Zeit wird das Körpergewebe gegenüber Schilddrüsenhormon empfindlich. Das HVL-Schilddrüsen-Regulationssystem wird ebenfalls zudiesem Zeitpunkt reaktionsbereit.Die Schilddrüse ist bis zu einem gewissen Grad selbstdifferenzierungsfähig; sie wird auch dann morphologisch ausdifferenziert — wenn auch verzögert und nicht in vollem Umfang —, wenn genügend exogenes Thyroxin vorhanden ist, um die Entwicklung weiterzuführen, und beim Fehlen der Hypophyse.Die Sekret-Produktion und -Speicherung in den -Zellen des HVL erfolgt auch dann, wenn genügend exogenes Thyroxin die TSH-Produktion überflüssig macht.Dinitrophenol dämpft die Aktivität der Schilddrüse, ohne daß die -Zellen des HVL hypertrophieren. Eine Hemmung des Rückkopplungsmechanismus ist wahrscheinlich.Natriumthiocyanat, Methylthiouracil und Kaliumperchlorat bewirken Kropfbildung und eine starke Hypertrophie der -Zellen des HVL. Dabei entsprechen die durch diese drei Substanzen verursachten Veränderungen der -Zellen nicht dem jeweiligen Aktivitätsgrad der Schilddrüse. So ist z. B. nach Thiocyanat-Behandlung die Schilddrüsenhemmung am stärksten, die Kernvergrößerung der -Zellen aber am geringsten. Natriumthiocyanat, Methylthiouracil und Kaliumperchlorat hemmen demnach nicht nur die Hormonproduktion der Schilddrüse unterschiedlich, sie greifen außerdem noch störend in das Regulationssystem ein: entweder durch Veränderung des Schilddrüsenhormons selbst oder des Rückkopplungsmechanismus.

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Summary The normal development of the thyroid gland and the pituitary during larval development and metamorphosis of the clawed toad Xenopus laevis Daudin and the changes of the anterior pituitary and the thyroid after treatment with dinitrophenol, thiocyanate, thiouracil, perchlorate and exceeding thyroxine were investigated.The capacity of self-differentiation and the effect of exogenous thyroxine on the thyroid were pointed out in hypophysectomized animals.Thyroid and TSH-producing cells (-cells) of the anterior pituitary start functioning at the beginning of the hindlimb-growth. At the same time the body tissue gets sensitive to thyroid-hormone and the anterior pituitary-thyroid-regulating system shows first signs of reactivity.The thyroid is able to selfdifferentiate up to a certain degree. The gland morphologically differentiates — though retarded and not to the full extent —, even if either sufficient exogenous thyroxine is available to proceed the development or the pituitary is lacking.The -cells of the anterior pituitary synthesize and store hormone, even if sufficient exogenous thyroxine makes the TSH-production unnecessary.Dinitrophenol restrains the activity of the thyroid without causing hypertrophy of the -cells of the anterior pituitary. An inhibition of the feed-back mechanism seems to be possible.Thiocyanate, thiouracil and perchlorate cause goitre and a strong hypertrophy of the -cells of the anterior pituitary. Considering the effects of these three substances, the changes of the -cells, caused by the lack of thyroid hormone, do not correspond with the respective activity of the thyroid. The inhibition of the thyroid is strongest after treatment with thiocyanate, but in this case the increase of the nuclear diameter of the -cells is smallest. Therefore thiocyanate, thiouracil and perchlorate do not only inhibit the hormone production of the thyroid. They also interfere with the regulation system in a different manner: they either modify the thyroid hormone itself or interfere with the feed-back mechanism.

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Dissertation der Naturwissenschaftlichen Fakultät der J. W. Goethe-Universität in Frankfurt a. M.Meinem Lehrer, Herrn Prof. Dr.H. Giersberg, danke ich für die Anregung dieser Arbeit, Herrn Prof. Dr. M. Lindauer für die weitere Überlassung des Arbeitsplatzes. Besonderen Dank schulde ich den Herren Prof. Dr. W. Hanke und Dr. F.-W. Pehlemann für jederzeit hilfreichen Rat.     

Effect of Long-Lasting Diabetes Mellitus on Rat and Human Brain Monoamines

Experimental alloxan- or streptozotocin-produced diabetes in rats was accompanied by an increase in the levels of norepinephrine, dopamine, and serotonin, whereas the contents of metabolites, i.e., 5-hydroxyindoleacetic acid and homovanillic acid, in the whole brain gradually decreased with the duration of diabetes. Among the striatum, thalamus, and hypothalamus of alloxan diabetic rats, monoamine alterations were observed only in the hypothalamus; after 1 week an increase of norepinephrine content and after 13 weeks an increase of norepinephrine and dopamine contents were found. Tissues of 11 brain regions of 10 diabetic and 12 control patients post mortem were investigated for monoamine concentrations. Patients were all male, of similar age and interval between death and autopsy. Diabetic patients had an increase in the content of serotonin in the medial and lateral hypothalamus. The content of dopamine increased in the medial hypothalamus, putamen, and medial and lateral pallidus. In diabetic patients, the content of norepinephrine increased in the lateral pallidus and decreased in the nucleus accumbens and claustrum. Thus, it seems that diabetes mellitus in rats, as well as in humans is associated with regionally specific changes in brain monoamines.     

Growth retarding effects of paclobutrazol and RSW 0411 on Granny Smith and Fuji apple trees

The growth retardants paclobutrazol (β-[(4-chlorophenyl)methyl]-α-(1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol) and RSW 0411 (β-(cyclohexyl methylene)-α-(1,1-dimethylethyl)-1H-1,2,4-triazole-1-ethanol) were tested on two-year-old trees of Granny Smith and Fuji apple. RSW 0411 at 100 mg/L did not cause any growth reduction in Granny Smith, while 500 and 1000 mg/L significantly reduced growth below that of the control between 27 and 40 days after application. Paclobutrazol at 100 mg/L had significantly reduced shoot growth between 27 and 55 days after application, and 1000 mg/L reduced shoot growth between 27 and 82 days after application. By 100 days after application, there were no longer differences between treatments. Shoot growth on Fuji trees was reduced below that of the control as follows: between 14 and 27 days following a single application of 500 mg/L RSW 0411; between 27 and 55 days following two applications; between 14 and 72 days following three applications; and between 14 and 82 days following four applications. Treatments were applied 14 days apart. Paclobutrazol was a more active growth retardant than RSW 0411 at the same rate, and the growth-retarding effects of RSW 0411 were short-lived.     

A birefringence study of changes in myosin orientation during relaxation of skinned muscle fibers induced by photolytic ATP release.

The birefringence of isolated skinned fibers from rabbit psoas muscle was measured continuously during relaxation from rigor produced by photolysis of caged ATP at sarcomere length 2.8-2.9 microns, ionic strength 0.1 M, 15 degrees C. Birefringence, the difference in refractive index between light components polarized parallel and perpendicular to the fiber axis, depends on the average degree of alignment of the myosin head domain with the fiber axis. After ATP release birefringence increased by 5.8 +/- 0.7% (mean +/- SE, n = 6) with two temporal components. A small fast component had an amplitude of 0.9 +/- 0.2% and rate constant of 63 s-1. By the completion of this component, the instantaneous stiffness had decreased to about half the rigor value, and the force response to a step stretch showed a rapid (approximately 1000 s-1) recovery phase. Subsequently a large slow birefringence component with rate constant 5.1 s-1 accompanied isometric force relaxation. Inorganic phosphate (10 mM) did not affect the fast birefringence component but accelerated the slow component and force relaxation. The fast birefringence component was probably caused by formation of myosin.ATP or myosin.ADP.Pi states that are weakly bound to actin. The average myosin head orientation at the end of this component is slightly more parallel to the fiber axis than in rigor.     

Gibberellic acid induces cytoplasmic acidification in maize coleoptiles

Indole-3-acetic acid (IAA), fusicoccin and weak acids all lower the cytoplasmic pH (pH_i) and induce elongation growth of maize (Zea mays L.) coleoptiles. Gibberellic acid (GA₃) also induces elongation growth and we have used confocal laser scanning microscopy to study the effects of GA₃ on pH_i employing the pH-indicator dyes, 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein and carboxy-semi-naphthorhodafluor-1. We confirm that GA₃ induces growth significantly in light-grown but only slightly or not at all in dark-grown coleoptiles. The growth induced by IAA treatment was similar in light- and dark-grown coleoptiles. The pH_i decreased by up to 0.6 units during the first 7 min of GA₃ or IAA treatment of both light- and dark-grown coleoptiles. Gibberellic acid inhibited IAA-induced growth of dark-grown coleoptiles. Hence, in dark-grown coleoptiles GA₃ may activate either directly or indirectly reactions that interfere with the signalling pathway leading to elongation growth. The possible role of pH_i in growth is discussed.Abbreviations ABA abscisic acid - AM acetoxymethyl ester - BCECF 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein - [Ca²⁺]_i cytoplasmic free calcium - GA_(n) gibberellin A_(n) - GA₃ gibberellic acid - IAA indole-3-acetic acid - PGR plant growth regulator - pH_i cytoplasmic pH - Pipes piperazine-N,N-bis[2-ethanesulfonic acid] - Snarf-1 carboxy-semi-naphthorhodafluor-1 We thank Dr R. King (CSIRO, Canberra) for providing the GA₁ and T. Phillips for processing the photographic material. H.R. Irving was supported by an Australian Research Council Research Fellowship and the work was supported by an Australian Research Council grant.     

Distribution of the enantiomers of hydroxychloroquine and its metabolites in ocular tissues of the rabbit after oral administration of racemic-hydroxychloroquine

The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc.     

The positions of 12 simple sequence repeat markers relative to reference loci on mouse Chromosome 16

The genetic map positions of 12 simple sequence repeat (SSR) markers spanning mouse Chromosome (Chr) 16 were determined relative to reference markers on that chromosome. Interval mapping data were obtained with a panel of DNAs from two intersubspecific backcrosses. All but one of the markers were typed by use of nonradioactive polymerase chain reaction (PCR) products analyzed on agarose gels. The marker order was determined to be Prm-1, D16Mit9, Igl-1, D16Mit29, D16Mit1/D16Mit2, Smst, D16Mit4, D16Mit11, Gap43, D16Mit14, D16Mit30, D16Mit5, Pit-1, D16Mit27, D16H21S16 (formerly D21S16h), D16Mit19, App, D16Mit7, Sod-1. Two of these markers mapped to the known human Chr 21 (HSA21)/Chr 16 conserved linkage group. Nine additional SSR markers could not be typed because they were not polymorophic (four markers), did not amplify MOLD/Rk DNA (three markers), or failed to give PCR products under a range of conditions (two markers). A subset of the most robust SSRs provide a useful marker set for the analysis of previously unmapped crosses.