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.     

Tyrant flycatchers coming out in the open: phylogeny and ecological radiation of Tyrannidae (Aves, Passeriformes)

Tyrant flycatchers constitute a substantial component of the land bird fauna in all South American habitats. Past interpretations of the morphological and ecological evolution in the group have been hampered by the lack of a well‐resolved hypothesis of their phylogenetic interrelationships. Here, we present a well‐resolved phylogeny based on DNA sequences from three nuclear introns for 128 taxa. Our results confirm much of the overall picture of Tyrannidae relationships, and also identify several novel relationships. The genera Onychorhynchus, Myiobius and Terenotriccus are placed outside Tyrannidae and may be more closely related to Tityridae. Tyrannidae consists of two main lineages. An expanded pipromorphine clade includes flatbills, tody‐tyrants and antpipits, and also Phylloscartes and Pogonotriccus. The spadebills, Neopipo and Tachuris are their closest relatives. The remainder of the tyrant flycatchers forms a well‐supported clade, subdivided in two large subclades, which differ consistently in foraging behaviour, the perch‐gleaning elaeniines and the sallying myiarchines, tyrannines and fluvicolines. A third clade is formed by the genera Myiotriccus, Pyrrhomyias, Hirundinea and three species currently placed in Myiophobus. Ancestral habitat reconstruction and divergence date estimation suggest that early divergence events in Tyrannida took place in a humid forest environment during the Oligocene. Large‐scale diversification in open habitats is confined to the clade consisting of the elaeniines, myiarchines, tyrannines and fluvicolines. This radiation correlates in time to the expansion of semi‐open and open habitats from the mid‐Miocene (c. 15 Mya) onwards. The pipromorphine, elaeniine and myiarchine–tyrannine–fluvicoline clades each employ distinct foraging strategies (upward striking, perch‐gleaning and sallying, respectively), but the degree of diversity in morphology and microhabitat exploitation is markedly different between these clades. While the pipromorphines and elaeniines each are remarkably homogenous in morphology and exploit a restricted range of microhabitats, the myiarchine–tyrannine–fluvicoline clade is more diverse in these respects. This greater ecological diversity, especially as manifested in their success in colonizing a wider spectrum of open habitats, appears to be connected to a greater adaptive flexibility of the search‐and‐sally foraging behaviour.     

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.     

Analysis of a glucose-containing tetrasaccharide by high-performance liquid affinity chromatography

In the present work we have explored conditions for using a pulsed amperometric detector for on-line analysis of oligosaccharides eluted from a high-performance liquid affinity chromatography column. A monoclonal antibody that specifically binds a glucose-containing oligosaccharide is coupled to a SelectiSphere-10-activated tresyl column. The system is eluted isocratically and easily detects 10 ng of the oligosaccharide with a linear response up to 250 ng. Analysis of both serum and urine samples from normal individuals and patients with acute pancreatitis gives a single retarded peak with a retention time identical to that of authentic (Glc)4. Retarded material pooled from several analyses of urine was positively identified as (Glc)4 by GC-MS analysis. As this method requires little cleanup and no chemical derivitization of the sample and is performed rapidly (less than 20 min) at sensitivities of at least 10 micrograms/liter in biological fluids, it represents a substantial improvement over previous GC-MS, radioimmunoassay, and enzyme-linked immunoadsorbent assay methods used to determine (Glc)4.     

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.