Robert W. K. Lee

Mutagenic activities of selected nitrofluoranthene derivatives in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6

The mutagenic activities of novel nitrofluoranthene derivatives in Salmonella strains TA98, TA98NR and TA98/1,8-DNP6 (with and without S9 addition) are given. These derivatives were produced from the reactions of fluoranthene (FL) and its directly mutagenic 2- and 3-nitro derivatives with covalent dinitrogen pentoxide (N2O5) in CCl4 solution at ambient temperature. The influence of the addition of a nitro group on the observed activity of the resulting di- and tri-nitrofluoranthenes is discussed.

Metabolic fate of alanine in an insect Manduca sexta: effects of starvation and parasitism

The fate of [3-13C]alanine administered to last instar larvae of an insect Manduca sexta was investigated in vivo by 13C-NMR spectroscopy. Following injection of the isotopically substituted substrate and conversion to [3-13C]pyruvate 13C was principally incorporated into C2, C3 and C4 of glutamate and glutamine in unparasitized ad libitum-fed larvae, insects starved 48 hr prior to injection and larvae parasitized by the insect parasite Cotesia congregata. Selective labeling at C2 and C3 of glutamate/glutamine resulted from carboxylation of [3-13C]pyruvate to [2,3-13C]oxaloacetate catalyzed by pyruvate carboxylase, randomization of the label in fumarate, and synthesis of glutamate and glutamine after condensation with acetyl CoA to [2 proR,3-13C]citrate. In contrast, enrichment at C4 of glutamate and glutamine resulted from oxidation [3-13C]pyruvate to [2-13C]acetyl CoA catalyzed by pyruvate dehydrogenase followed by condensation with oxaloacetate. The ratio of enrichment (C2 + C3): C4 provided a measure of the relative contributions of the pyruvate dehydrogenase and pyruvate carboxylase catalyzed pathways of substrate utilization by the tricarboxylic acid cycle. The mean ratio was 0.6 and 0.7 in control and parasitized larvae, respectively, and 2.4 in starved insects. The latter result demonstrated that substrate utilization by the TCA cycle was markedly altered by starvation. In addition, the rate of labeled alanine metabolism was significantly reduced by starvation. The concentrations of glutamate and glutamine in the blood (hemolymph) were similar in all three groups of insects. No evidence for gluconeogenesis was observed in any group. Starved larvae incorporated label into C6 of glucose and trehalose but no complementary enrichment at C1 was observed. This result was consistent with the activity of the non-oxidative phase of the pentose phosphate pathway during which labeled glyceraldehyde-3-phosphate arising from [3-13C]alanine reacts with sedoheptulose-7-phosphate yielding erythrose-4-phosphate and [6-13C]fructose-6-phosphate catalyzed by transaldolase. Specifically labeled fructose-6-phosphate then gives rise to glucose and trehalose labeled at C6. Preliminary analysis of the hemolymph of starved insects indicated the presence of several hexose phosphates labeled at C6. The hemolymph level of trehalose was significantly reduced in both starved and parasitized insects. Lipogenesis from [3-13C]alanine was evident in unparasitized control larvae but was absent in parasitized and starved insects. The pattern of labeling in fatty acid was consistent with de novo pathway utilizing [2-13C]acetyl CoA derived by oxidation of [3-13C]alanine.

Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.

Glucose metabolism in an insect Manduca sexta and effects of parasitism

The metabolism of [1-13C]glucose was examined during the last larval stadium of an insect Manduca sexta (Lepidoptera: Sphingidae) parasitized by Cotesia congregata (Hymenoptera: Braconidae). Following injection, the isotopically substituted glucose was metabolized at a significantly lower rate by parasitized larvae than by normal, control insects. 13C enrichment was principally observed in [1-13C]trehalose in both groups. Randomization of the label at the triose phosphate step was evidenced by incorporation of 13C into C6 of trehalose. Parasitized and control larvae both synthesized [1,6-13C]glycogen but the relative amount of label observed in parasitized larvae was greater. The ratio of C6/C1 enrichment in trehalose and glycogen was significantly less in parasitized larvae. The rate of labelled trehalose and glycogen synthesis was relatively high when compared with the estimated rate of glycolytic glucose oxidation, and the difference in C6/C1 enrichment ratio between normal and parasitized insects was, therefore, not reflective of a difference in the rate of substrate cycling, but rather, was due to the increased synthesis of [1-13C]glycogen in parasitized larvae when compared with controls. Inhibition of glycolysis by administration of iodoacetate to normal larvae resulted in an increase in the incorporation of 13C into glycogen relative to glucose metabolized, suggesting that inhibition of glycolysis may be responsible for the higher level of glycogen synthesis observed in parasitized insects. In control larvae, significant 13C enrichment from [1-13C]glucose was observed in fat, but no evidence of lipogenesis was observed in parasitized insects. Iodoacetate had no observable effects on the relative amount of 13C incorporated into fat. Malonic acid and cyanide resulted in accumulation of 13C from [1-13C]glucose in several TCA cycle intermediates of normal larvae, but had little effect on the relative enrichments of trehalose, glycogen and fat.

In vivo 31P NMR spectrum of Hymenolepis diminuta and its change on short-term exposure to mebendazole

The 31P NMR spectrum of the adult tapeworm, Hymenolepis diminuta, at 37 degrees C during perfusion with physiological saline was composed of 10 peaks. Based on chemical shifts and analysis of worm extracts, the phosphorus components included glucose-6-phosphate, fructose-6-phosphate, phosphorylcholine, glycerophosphoryl choline and -ethanolamine, nucleotide monophosphate-diphosphate and -triphosphate, nicotinamide adenine dinucleotide and uridine diphosphate glucose. The mean level of nucleotide triphosphate was 0.86 nmol (mg fresh weight)-1 and the nucleotide triphosphate/-diphosphate ratio 3.9. Based on the nucleotide triphosphate level, worms were viable for at least 3 h and the intracellular pH was maintained constant at approximately 6.7. Short-term exposure to mebendazole perfused at 11 or 27 microM solubilized in physiological saline containing 0.5% Tween 80 or 0.1% dimethyl sulphoxide had little effect on the nucleotide triphosphate level. Some cytological changes, however, were evident following perfusion of mebendazole. In contrast, exposure to 2,4-dinitrophenol caused a rapid decline in nucleotide triphosphate level. It was concluded that mebendazole does not exert its primary effect on oxidative phosphorylation.

Sensitivity enhancement for 13C-1H 2D J-resolved NMR using a recycled-flow method

Abstract A recycled-flow NMR system is evaluated for application to two-dimensional 13C-1H J-resolved experiments. For proper choice of measurement conditions, it is demonstrated that spectral sensitivity enhancement is inversely proportional to 1 − exp ( −t a T 1 ) , where ta is the average experiment time and T1 the spin-lattice relaxation time. The enhancement factor of flow over static measurements for slowly relaxing carbon nuclei (T1 about 50–85 s) is between 3.5 and 5.0 and is between 1.5 and 2.5 for rapidly relaxing carbons (T1 about 5–10 s) in α-pinene. Choice of flow rate, pulse repetition time, evolution period, and spectral resolution, as well as the value of T1s, all affect experimental sensitivity enhancement for 2D flow NMR spectra.

Phosphoglycerides and derivatives in Taenia crassiceps examined by 31P NMR spectroscopy

Examination of the larval stage of the tapeworm, Taenia crassiceps, by 31P NMR spectroscopy revealed the presence of a major phosphoglyceride component. However, using saturation transfer, no exchange between glycerophosphorylcholine and phosphoglyceride or any other NMR-detectable phosphorus metabolites was detected.

31P NMR spectra of mosquito larvae parasitized with Romanomermis culicivorax

Abstract The energy status of Romanomermis culicivorax-parasitized and control Culex pipiens larvae was studied in vivo with 31P nuclear magnetic resonance (NMR) at 4–6°C. Peak assignments were made for inorganic phosphorus, phosphoarginine, AMP, ADP, and ATP. The mean ATP ADP ratios were 4.45 for control and 4.02 and 2.94 for parasitized mosquito larvae with 1.3 and 2.3 parasites per host, respectively. The ATP ADP ratios for control mosquito larvae reared with a pH of 7 and 4.5 were not significantly different. R. culicivorax parasitism had no significant effect on the ATP level in whole mosquito larvae. The mean ATP concentrations were 9.86 and 11.64 nmol/mg dry weight mosquito for control and parasitized larvae, respectively. R. culicivorax postparasites (10 days post-infection) were shown to have a very weak 31P NMR signal and it was concluded that the parasites contribute little to the NMR spectra of infected mosquito larvae. Enzymatic analysis of perchloric acid-extracted control mosquito larvae yielded 9.3 nmol ATP/mg dry wt and 2.6 nmol ATP/mg dry weight for R. culicivorax postparasites at 10 days post-infection.

THE 2-ZINC INSULIN HEXAMER IS A CALCIUM-BINDING PROTEIN

Publisher Summary This chapter outlines X-ray diffraction structures for the 3Cd-insulin hexamer (at 1.9 A spacing) the 5Pb-insulin hexamer (at 2.5 A spacing) and the metal-free insulin hexamer (at 2.5 A spacing). Most spectra shown are the average of 200 to 400 transients collected over a time period of 20 to 40 m. The electron density section taken through the His(B10) site indicates a distorted octahedral ligand field, consisting of three histidyl imidazolyl nitrogens and three water molecules. Other changes in the aromatic region occur upon assembly of the metal-substituted insulin hexamers. The changes because of assembly involve perturbation of the environments of Phe and Tyr residues as the dimer-dimer interfaces are formed. Binding of metal ions to the Glu(B13) site stabilizes the insulin hexamer and causes a conformational perturbation that influences the microenvironments of distant residues along the dimer-dimer interfaces.

1H FT NMR STUDIES OF THE Co3+-SUBSTITUTED HEXAMER: CHARACTERIZATION OF METAL ION BINDING TO THE GLU(B13) SITE

Publisher Summary This chapter presents results of the 1 H FT Nuclear Magnetic Resonance (NMR) studies of the Co 3+ -substituted hexamer for characterization of metal ion binding to the Glu(B13) site. In vivo , within the insulin secretory granules, crystalline insulin hexamers are formed and stabilized by the binding of Zn 2+ and Ca 2+ . These metal ions help hold the hexamer together by bonding across dimers. The Glu(B13) site has also been identified as a calcium binding site. When insulin is released into the bloodstream, the insulin crystals dissolve and hexamers must dissociate to monomers for insulin to become active. In this chapter, the 1 H NMR spectra of cobalt-substituted insulin, In 6 -Co 2 3+ and cobalt-substituted insulin derivatives, with Glu-(B13) bound calcium, In 6 Co 2 3+ , Ca 1 2+ , cadmium In 6 Co 2 3+ , Cd 1 2+ or lead In 6 Co 2 3+ Pb 3 2+ are presented. The resonance for His(B10) is easily identified (for once) as the very broad resonance at 8.53 ppm. When tripositive cobalt binds to the His(B10) sites, the His(B10) signals are shifted downfield to an uncommon extent and the rigidly bound cobalt increases the linewidth resulting in a broad, symmetrical peak.