Change in electron and spin density upon electron transfer to haem

Haems are the cofactors of cytochromes and important catalysts of biological electron transfer. They are composed of a planar porphyrin structure with iron coordinated at the centre. It is known from spectroscopy that ferric low-spin haem has one unpaired electron at the iron, and that this spin is paired as the haem receives an electron upon reduction (I. Bertini, C. Luchinat, NMR of Paramagnetic Molecules in Biological Systems, Benjamin/Cummins Publ. Co., Menlo Park, CA, 1986, pp. 165-170; H.M. Goff, in: A.B.P. Lever, H.B. Gray (Eds.), Iron Porphyrins, Part I, Addison-Wesley Publ. Co., Reading, MA, 1983, pp. 237-281; G. Palmer, in: A.B.P. Lever, H.B. Gray (Eds.), Iron Porphyrins, Part II, Addison-Wesley Publ. Co., Reading, MA, 1983, pp. 43-88). Here we show by quantum chemical calculations on a haem a model that upon reduction the spin pairing at the iron is accompanied by effective delocalisation of electrons from the iron towards the periphery of the porphyrin ring, including its substituents. The change of charge of the iron atom is only approx. 0.1 electrons, despite the unit difference in formal oxidation state. Extensive charge delocalisation on reduction is important in order for the haem to be accommodated in the low dielectric of a protein, and may have impact on the distance dependence of the rates of electron transfer. The lost individuality of the electron added to the haem on reduction is another example of the importance of quantum mechanical effects in biological systems.     

Priors for the Bayesian star paradox

We show that the Bayesian star paradox, first proved mathematically by Steel and Matsen for a specific class of prior distributions, occurs in a wider context including less regular, possibly discontinuous, prior distributions.     

Direct induction of lymphocyte killing by calcium ionophore and phorbol ester treatment

To analyze transduction mechanisms in human lymphocyte killing, intracellular Ca2+ levels were increased by ionophore A23187 treatment and protein kinase C activated by phorbol ester 12-O-tetradecanoylphorbol-acetate (TPA). Drugs were tested either alone or in combinations on effector cells active in natural, antibody-dependent, and lectin-dependent killing. TPA suppressed killing in all systems at 100 ng/ml whereas A23187 was only suppressive for NK killing at concentrations higher than 0.1 microM. TPA combined with A23187, above 10 ng/ml and 0.5 microM, respectively, induced killing of all tested target cell lines with a slower kinetic than NK killing of K562 cells. Drug-induced killing did not increase optimal lectin and antibody-dependent killing and was demonstrated most easily on NK-resistant target cell lines. Fractionation of effector lymphocytes into NK cell-depleted, T3-positive and NK cell-enriched, T3-negative cells demonstrated that similar levels of TPA/A23187-dependent killing could be induced in both fractions. It is concluded that TPA/A23187 induce normal lymphocytes to nonselective killing of different target cells in similarity to the triggering effect these drugs have in many other cell systems. Whether the induced killing is representative of NK killing is discussed in relation to the presence of other potential effector cells and effector molecules in peripheral blood lymphocytes.     

Differential effect on inositol-phospholipid hydrolysis, cytosolic-free Ca2+ concentration, protein kinase C activity and protein phosphorylation of 1-oleoyl-2-acetyl-sn-glycerol growth-stimulated ascites tumor cells

This study shows that the membrane-permeable stereospecific 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the analog of the natural 1,2-diacylglycerol (DAG), can stimulate the growth of ascites tumor cells. OAG can fully replace high serum concentrations in the culture medium and stimulates DNA synthesis in a dose-dependent manner. Investigation of the protein kinase C (PKC) isolated from a Triton extract of a 100,000g membrane pellet revealed that OAG can directly activate this enzyme. Concomitantly the phosphorylation of several cytosolic proteins with the molecular weights of 26, 33, 49, 55, 64, and 90 kDa is observed which is also found in serum-stimulated cells. Since DAG as a second messenger molecule originates from the hydrolysis of phosphoinositides we have investigated the metabolism of these lipids after labeling the cells with [3H]inositol. In detail, we have measured the amount of radioactive inositol trisphosphate (IP3) and the phosphodiesterase hydrolyzing phosphatidylinositol-4,5-bisphosphate (PIP2). The decreased radioactivity level of IP3 in OAG-stimulated cells as compared to non-growing cells (1-2% serum) indicates a feedback regulation of PIP2 hydrolysis which is substantiated by a profound reduction of PIP2-specific phospholipase C activity. The reduced IP3 formation has apparently no inhibitory effect on the cytoplasmic free Ca2+ concentration of OAG-stimulated cells, suggesting that the Ca2+ release is not directly correlated to the amount of IP3, which is also demonstrated for the non-growing cells. These data indicate that OAG apparently has a duel effect on the inositol phospholipid-mediated signal transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Transformations of Halogenated Aromatic Aldehydes by Metabolically Stable Anaerobic Enrichment Cultures

Metabolically stable enrichment cultures of anaerobic bacteria obtained by elective enrichment of sediment samples from the Baltic Sea and Gulf of Bothnia have been used to study the oxidation and reduction of the aldehyde group of various halogenated aromatic aldehydes. During the transformation of 5- and 6-chlorovanillin, 6-bromovanillin, 3-chloro-4-hydroxybenzaldehyde, 3,5-dichloro-4-hydroxybenzaldehyde, and 3,5-dibromo-4-hydroxybenzaldehyde, it was shown that synthesis of the corresponding carboxylic acids, which were the principal metabolites, was invariably accompanied by partial reduction of the aldehyde to a hydroxymethyl group in yields of between 3 and 30%. Complete reduction to a methyl group was observed with some of the halogenated vanillins, but to an extremely limited extent with the halogenated 4-hydroxybenzaldehydes. One consortium produced both the hydroxymethyl and methyl compounds from both 5- and 6-chlorovanillin: it was therefore assumed that the methyl compound was the ultimate reduction product. On the basis of the kinetics of formation of the metabolites, it was concluded that the oxidation and reduction reactions were mechanistically related. In addition to these oxidations and reductions, dehalogenation was observed with one of the consortia. In contrast to the transformations of 5- and 6-chlorovanillin, which produced chlorinated methylcatechols, the corresponding compounds were not observed with 5- and 6-bromovanillin: the former was debrominated, forming 4-methylcatechol, whereas the latter produced 6-bromovanillyl alcohol without demethylation. Similarly, although 3-chloro-4-hydroxybenzaldehyde formed the chlorinated carboxylic acid and the benzyl alcohol, the 3-bromo compound was debrominated with formation of 4-hydroxybenzoic acid and, ultimately, phenol. On prolonged incubation, the halogenated carboxylic acids were generally decarboxylated, so that the final products from these substrates were halogenated catechols or phenols. Reductive processes of the type revealed in this study might therefore plausibly occur in the environment during anaerobic transformation of halogenated aromatic aldehydes containing hydroxyl and/or methoxyl groups.     

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71

Summary Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

Fractionation and characterization of rapidly phosphorylated nuclear proteins in salivary gland cells of Chironomus tentans

Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with ³²P_i. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of ³²P) and not sedimentable (supernatant) material. About 90% of ³²P in the supernatant fraction was sedimentable at 100,000 g (disperse chromatin). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of ³²P. The ³²P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the ³²P-labeled nuclear proteins into 12 major bands in the M_r range of 12,000–120,000. The incorporation of ³²P into most bands reached a steady-state within 5–10 min of incubation with ³²P_i and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an M_r 95,000 phosphoprotein. Two of the fastest moving ³²P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving ³²P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.