Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase

The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD⁺ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD⁺. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD⁺ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase_TOL with dioxygen and thus present a solution toward conflicting requirements.     

ΔF508 testing of the DNA bank of the Royal Manchester Children's Hospital

Summary Details of haplotype and ΔF508 status from various populations represented in the cystic fibrosis (CF) DNA bank of the Royal Manchester Children's Hospital are provided, together with information on the association of genotype and clinical status. Clinical details and DNA analyses from native English in the North-West and South-West of England (Bath), from Lancashire Pakistani families and from Afrikaans Namibian families are compared. A 78.5% incidence of ΔF508 has been found in English families. Compound heterozygotes with CF and only one ΔF508 gene have an increased likelihood of having milder disease, with lessPseudomonas isolated from sputum and relatively more showing either no regular respiratory pathogens or colonisation withStaphylococcus. There is also a relative increase in meconium ileus in these compound heterozygotes. The diagnosis of CF may be in doubt in some subjects negative for ΔF508. Some of the Bath families have unusual haplotypes for an English population and a compound heterozygote ΔF508/ΔI507 has been found. There is evidence from metD analysis of the founder effect in the Afrikaans Namibian families, who have a high ΔF508 incidence.     

Unimpaired ability to generate adherent lymphokine-activated killer (A-LAK) cells in patients with primary or metastatic liver tumors

Summary Adherent lymphokine-activated killer cells (A-LAK cells) obtained from human peripheral blood mononuclear cells represent a population of potent antitumor effectors enriched in interleukin-2(IL-2)-activated natural killer cells. This study shows that A-LAK cells can be successfully generated from the blood of patients with liver cancer not treated with adjuvant chemotherapy or irradiation. Mononuclear cells were isolated from the blood of 33 patients with liver tumors (6 benign, 10 primary malignant, 17 metastatic) at the time of liver resection. A-LAK cells were separated by adherence to plastic following activation of peripheral blood mononuclear cells in 1000 U/ml recombinant IL-2. A-LAK cells (enriched up to 92% in CD3^–CD56⁺ cells) showed better subsequent expansion and two to six times higher antitumor cytotoxicity per cell than unseparated LAK cells cultured under the same conditions. The ability to generate A-LAK cells with superior in vitro cytotoxicity from the blood of most patients with liver cancer indicates that adoptive cellular immunotherapy may be a feasible and new way of treatment for primary and secondary hepatic neoplasms in man.     

Effects of pentachlorophenol and some of its known and possible metabolites on different species of bacteria

The antibacterial activity of pentachlorophenol and 35 of its known or possible metabolites against 30 different species of bacteria was tested. In comparison with pentachlorophenol, no increase of inhibitory activity was found for any of the chlorinated anisoles tested (except for pentachloroanisole against Streptomyces spp.), 2-chlorophenol, 2,6-dichlorophenol, 2,3,6- and 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, tetrachloro-1,4- and -1,3-benzenediol (except for the 1,3-isomer against Streptomyces spp.), tetrachloro-1,3-dimethoxybenzene, and tetrachloro-1,3-benzenediol diacetate. Two chlorophenols, five dichlorophenols, four trichlorophenols, two tetrachlorophenols, and tetrachloro-1,2-benzenediol were more active than pentachlorophenol against some, but not all, of the strains tested.     

Sex pheromone of the female currant borer, Synanthedon tipuliformis: Identification and field evaluation

(E,Z)-2,13-octadecadien-1-ol acetate and (Z)-13-octadecen-1-ol acetate have been identified from the ovipositor extracts of the currant borer, Synanthedon tipuliformis (Clerck), by capillary GC, and the structures were verified by GC-MS. The diene/monoene ratio in the extract was found to be 93:7. Mixtures of these two compounds as well as the diene alone attracted conspecific males in the field.

---

Zusammenfassung (E,Z)-2,13-Octadecadienylacetat und (Z)-13-Octadecenylacetat wurden in Ovipositor-Extrakten des Johannisbeerglasflüglers Synanthedon tipuliformis kapillar-gaschromatographisch nachgewiesen. Die Strukturen wurden mittels Gaschromatographisch-Massenspektrometrie bestätigt. Das Verhältnis Dien/Monoen im Extrakt betrug 93:7. Mischungen der beiden Komponenten wie auch (E,Z)-2,13-Octadecadienylacetat allein lockten im Freiland Männchen dieser Glasflüglerart an.

     

Histochemistry of complex carbohydrates in the skin of the pig snout, with special reference to eccrine glands

The histochemistry of carbohydrates has been studied in the skin of the pig snout with selected methods of light microscopy including peroxidase-labelled lectin-diaminobenzidine (PO-LT-DAB) procedures. In the snout skin the dark secretory cells and the luminal secretion of the eccrine glands contained considerable amounts of neutral glycoproteins, but only a very small amount of acidic ones. It was possible to demonstrate glycogen in the secretory cells of these glands. The other skin structures of the snout also showed positive reactions for complex carbohydrates. Most remarkable were stronger reactions of intercellular substances among the spinosum cells, particularly following the PO-LT-DAB procedures, which demonstrated such saccharide residues as beta-D-galactose and N-acetyl-D-glucosamine.