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.     

Identification and characterization of mutations responsible for a runaway replication phenotype of plasmid R1

Initiation of replication of the resistance plasmid R1 is carefully regulated by the two negatively acting factors, CopA and CopB. It is shown here that the temperature-dependent runaway-replication phenotype of an R1 plasmid mutant is caused by two point mutations in each of the promoters for the genes of these control factors. Expression of the two genes is affected in the following way: (1) one C-to-T transition in the putative -35 box of the copB-repA operon creates a two- to three-fold stronger promoter from which expression is temperature-dependent; (2) another C-to-T transition in a G + C-rich area immediately downstream from the -10 box of the copA promoter reduces expression of the copA gene three-fold. The phenotypic consequences of the two mutations are discussed in the light of the current model for R1 replication control.     

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.     

Cloning and expression in Escherichia coli of the gene for extracellular phospholipase A1 from Serratia liquefaciens.

From a genomic library of Serratia liquefaciens, a cloned DNA fragment comprising a two-gene operon was isolated and expressed in Escherichia coli. One of the gene products was identified as a phospholipase A1, and the enzyme was found to be excreted to the outer environment from S. liquefaciens as well as from E. coli. Both genes were sequenced, and the relationship between open reading frames in the DNA sequence and in vitro-expressed polypeptides was established. The length of the phospholipase polypeptide was found to be 319 amino acids. In the amino-terminal end of the coding sequence was a stretch of about 20 hydrophobic amino acids, but, in contrast to consensus signal peptides, no basic residues were present. The length of the second polypeptide was 227 amino acids. It was found that expression of the phospholipase gene in both E. coli and S. liquefaciens was growth phase regulated (late expression).     

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.     

Characterization of new gangliosides of the lactotetraose series in murine xenografts of a human glioma cell line

The major mono- and disialogangliosides of the extensively characterized established human glioma line D54MG were isolated and purified from subcutaneous solid xenografts grown in athymic (nu/nu) mice. Structural determination showed that they belonged to the lactotetraosylceramide series. The sialyllactotetraosylceramide contained 90% N-glycolyl- and 10% N-acetylneuraminic acid linked in an alpha 2-3 linkage (IV3NeuGc-LcOse4Cer, IV3NeuAc-LcOse4Cer). The disialogangliosides had a previously undescribed type of structure with sialic acids linked to the terminal galactose in an alpha 2-3 linkage and to N-acetylglucosamine in an alpha 2-6 linkage. Not only did species with NeuAc or NeuGc occur, but also species with mixtures of the two sialic acids, e.g. NeuAc and NeuGc. The schematic structures of the new disialogangliosides are (Formula:see text).     

Effect of Different Carbon Sources on the Ammonium Induction of Different Forms of NADP-Specific Glutamate Dehydrogenase in Chlorella sorokiniana Cells Cultured in the Light and Dark

The ammonium induction of the chloroplast-localized NADP-specific glutamate dehydrogenase (NADP-GDH) was shown not to be a light-dependent process per se in Chlorella sorokiniana. In the dark without exogenous organic substrates, the cells synthesized low levels of fully active NADP-GDH, provided endogenous starch reserves had not been depleted. When cells were supplied with exogenous acetate, the rate of induction of NADP-GDH activity per milliliter of culture in the dark was equal to or slightly greater than the rate observed under photosynthetic conditions without an organic carbon source. Glucose supported only a low rate of induction of NADP-GDH activity in the dark. Both acetate and glucose inhibited induction of enzyme activity in the light. The NADP-GDH holoenzyme had at least 7 different electrophoretic forms. These forms differed in net charge and/or molecular weight. Their difference in molecular weight was due to the presence of 2 subunits with similar antigenic properties but different molecular weights (M_r = 55,500 and 53,000; α-and β-subunits, respectively). Depending upon the cultural conditions and length of the induction period, a wide variation was observed in the α:β subunit ratio and in the numbers and sizes of the NADP-GDH holoenzymes.