Use of inversion spin transfer to monitor creatine kinase kinetics in rat skeletal muscle in vivo

A simple multipulse sequence has been used to monitor creatine kinase kinetics in rat skeletal muscle in vivo. Using these procedures, the forward (ATP synthesis) and reverse fluxes (phosphocreatine synthesis) have been calculated to be 8.98 +/- 0.6 and 10.7 +/- 0.8 mumoles/g wet wt/s (n = 5) respectively. These results suggest that in resting skeletal muscle most of the gamma ATP observed in 31P NMR spectra is cytosolic and rapidly exchanging with phosphocreatine. The high flux rates reflect the high catalytic capacity of creatine kinase in skeletal muscle.     

Differential processing of Asn-linked oligosaccharides on pituitary glycoprotein hormones: implications for biologic function

Luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone from pituitary and chorionic gonadotropin from placenta are a family of glycoproteins, each consisting of an and subunit. Within an animal species, the subunit of all four hormones contains the identical amino acid sequence, while each subunit is distinct and confers biologic specificity to the hormone dimer. Despite sharing common subunits, these hormones bear Asn-linked oligosaccharides which differ in structure. Whereas chorionic gonadotropin contains exclusively neutral and sialylated oligosaccharides, the pituitary hormones bear neutral, sialylated, sulfated, and sialylated/sulfated structures. The sulfated oligosaccharides are unique in structure and are more prevalent on certain pituitary hormones, indicating that the synthesis of these unusual oligosaccharides is tightly regulated. The differences in oligosaccharide structures in conjunction with the highly specific endocrine responses elicited by these hormones, suggest an important functional role for the oligosaccharides, such as metabolic clearance, control of hormone response, modulation of hormone potency, and/or intracellular sorting of hormones into separate secretory granules.     

Microbial fermentative preparation of L-[15N2]lysine and its tracer: application to serum amino acid kinetic studies

The microorganism Brevibacterium flavum 21129 has been used to produce multigram batches of L-[15N2]lysine of high purity and isotopic enrichment by supplementation of the growth medium with (15NH4)2SO4 of 98.0 atom% excess. The doubly 15N-labeled lysine can be detected at dilutions 10 times greater than singly labeled lysine when isotope dilution curves are analyzed by gas chromatography-mass spectrometry. This enhanced sensitivity permits kinetic measurements of plasma free-lysine isotope content over a 300-fold dilution during 6 h following a single oral bolus of 5 mg/kg body wt. This inexpensive preparation method lends itself to the production of highly useful biochemical compounds for kinetic studies of human nutrition.     

Neocarzinostatin chromophore: Presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride

Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C₁₂-subunit of the previously determined partial structure $\text{2a}$ (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C₁₂-substructure, whereas a parallel two-step reduction occurs on NaBH₄ treatment. Both reactions occur with rearrangement of the C₁₂-substructure and the implication for the mechanism of action of NCS-Chrom $\text{A}$ in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom $\text{A}$ as well as minor components $\text{B}$ and $\text{C}$ by two protons.     

New Regulatory Mutation Affecting Some of the Tryptophan Genes in Pseudomonas putida

Three indole analogues, 5-methylindole, 5-fluoroindole, and 7-methylindole, and the tryptophan analogue 5-fluorotryptophan were found to inhibit the growth of wild-type Pseudomonas putida. Mutants resistant to these analogues were obtained. Some of the 5-fluoroindole- and 5-fluorotryptophan-resistant strains exhibit an abnormality in the regulation of certain trp genes. These strains excrete anthranilate when grown in minimal medium in the presence or absence of the inhibitor. In these strains, the trpA, B, and D gene products, the first, second, and fourth enzymes of the tryptophan pathway, are produced in 20-fold excess over the normal wild-type level. The other enzymes of the pathway are unaffected. Exogenous tryptophan is still able to repress the expression of the trpABD cluster somewhat. Similarity between the 5-fluoroindole- and 5-fluorotryptophan-resistant strains suggests that the former compound becomes effective through conversion to the latter. Repression and derepression experiments with two anthranilate-excreting, 5-fluoroindole-resistant strains showed coordinate variation of the affected enzymes. The locus conferring resistance and excretion is not linked by transduction to any of the trp genes.     

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.     

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.