On the mechanism of the peroxidase-catalyzed oxygen-transfer reaction

We reported evidence that horseradish peroxidase (HRP) and chloroperoxidase (CPO) catalyze oxygen transfer from H2O2 to thioanisoles [Kobayashi, S., Nakano, M., Goto, T., Kimura, T., & Schaap, A. P. (1986) Biochem. Biophys. Res. Commun. 135, 166-171]. In the present paper, the reaction mechanism of this oxygen transfer is discussed. The oxidation of para-substituted thioanisoles by HRP compound II showed a large negative rho value of -1.46 vs. the sigma + parameter in a Hammett plot. These results are in accord with the formation of a cation radical intermediate in the rate-determining step. Hammett treatments for HRP- and CPO-dependent S-oxygenations did not provide unequivocal proofs to judge the reaction mechanism, because of the poor correlations for sigma + and sigma p parameters. Different behavior was found in kinetics and stereoselectivity between the two enzymes. Results in the present study and recent studies strongly suggested the formation of a cation radical intermediate. The oxygen atom would transfer by reaction of compound II and the cation radical intermediate. Although involvement of the cation radical was not confirmed in the CPO system, a similar mechanism was proposed for CPO.     

Cyclic-AMP binding to the cell surface during development of Dictyostelium discoideum

Abstract. Cell aggregation in Dictyostelium discoideum is a chemotactic process mediated by cyclic adenosine monophosphate (CAMP), which is detected by cell surface receptors. The cAMP signal is degraded by cAMP phosphodiesterase. The possibility that cAMP signals are also used for cell communication in the multicellular stages was studied by determining whether the cAMP receptors, which are essential for signal transduction, continue to function in these stages. During slug migration, the number of binding sites per cell decreases to about 15% of the maximum level acquired during aggregation. At the onset of fruiting body formation, a three- to Four-Fold increase in cAMP binding activity occurs. This increase coincides with an increase in cAMP phosphodiesterase. Both phenomena suggest that cell-cell communication mediated by cAMP is used during culmination. During both slug migration and early culmination, the prestalk cells exhibit about twice as much binding activity as the prespore cells.     

In vivo and in vitro analysis of structure-function relationships in ribosomal protein L25 from Saccharomyces cerevisiae

We have developed a combination of in vivo and in vitro methods which allows us to determine the effect of practically every structural change, deletions as well as point mutations, on various biological functions of a ribosomal protein (r-protein). We have used this approach to delineate the functional domains of r-protein L25 from Saccharomyces cerevisiae. By analysis of the intracellular distribution of fusion proteins carrying various portions of L25 linked to Escherichia coli beta-galactosidase we traced the nuclear localization signal(s) of L25 to the region encompassing the N-terminal 61 amino acids of the protein. On the other hand, using in vitro prepared fragments of L25 we located the domain responsible for its specific binding to 26S rRNA to the region between amino acids 61 and 135. In order to be able to analyze the effect of mutations in L25 on ribosome biogenesis and function in vivo we constructed a mutant yeast strain in which the chromosomal L25 gene is placed under control of the inducible yeast GAL promoter. Since this strain is unable to grow on glucose as a carbon source the L25 gene must be essential for cell viability. Growth on glucose can be restored by introduction of a wild-type L25 gene on a plasmid, demonstrating that under these conditions the cells are dependent upon an extrachromosomally supplied copy of the gene.     

Genome Analysis and Physiological Comparison of Alicycliphilus denitrificans Strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601^T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601^T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601^T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601^T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601^T. Genes involved in cyclohexanol degradation were only found in strain K601^T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.     

Malaria and urbanization in sub-Saharan Africa

There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.     

Functional Promiscuity of Gene Regulation by Serpentine Receptors in Dictyostelium discoideum

Serpentine receptors such as smoothened and frizzled play important roles in cell fate determination during animal development. In Dictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1 car3 double mutant cell lines that express equal levels of either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses and induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed. (i) Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. cAR1’s mediation of adenosine inhibition suggests that cAR1 normally mediates prespore gene induction. (ii) Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by differentiation-inducing factor (DIF) but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs cAR2 is expressed only in anterior cells, this could explain the so far puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2, and cAR3.Recent years have seen the discovery of critical roles in animal development for serpentine receptors, which are usually coupled to heterotrimeric G proteins. The insect sigaling peptides hedgehog and wingless and their mammalian counterparts sonic hedgehog, desert hedgehog, and indian hedgehog and the wnt factors control a multitude of inductive events during all stages of embryogenesis. The hedgehog signal is detected by two different serpentine receptors, smoothened (1, 40) and patched (21, 38), whereas the wingless or wnt signal is detected by the serpentine receptor D-frizzled-2 (3). In the social amoeba Dictyostelium discoideum, serpentine cyclic AMP (cAMP) receptors (cARs) control induction of cell differentiation during the entire course of development. Starving cells secrete cAMP pulses that induce chemotaxis and expression of genes required for the aggregation process. Cells aggregate to form mounds, which ultimately transform into fruiting structures that consist of a globular spore mass supported by a column of stalk cells. cAMP induces entry into the spore differentiation pathway as well as synthesis of a lipophilic factor, differentiation-inducing factor (DIF), which induces entry into the stalk differentiation pathway (see reference 5). At an early stage of development cAMP synergizes with DIF to induce prestalk genes, but later it becomes an inhibitor of stalk gene expression (2). cARs were shown previously to mediate induction of aggregative genes by cAMP pulses (20) as well as cAMP induction of prespore genes and repression of prestalk genes (31, 37). Remarkably, the target for the latter critical step in cell fate determination is glycogen synthase kinase 3 (GSK-3), a zeste white-3 homolog, which is the target for the effects of wingless and wnt in insects and vertebrates, respectively (7, 34).Four cARs, showing 54 to 69% amino acid identity, are expressed in a stage- and cell-type-specific manner. cAR1 is predominantly expressed before and during aggregation (18). cAR3 is expressed at late aggregation, and expression is later restricted to the prespore cell population (13, 44). cAR2 and cAR4 are both expressed exclusively in the prestalk cell population after aggregation (19, 30). cAR knockout cell lines were generated to examine the role of the individual cARs in Dictyostelium development. car1 null cells neither aggregate nor express developmental genes but can be triggered to express aggregative and postaggregative genes by stimulation with cAMP (37, 39). car3 null cells aggregate and develop normally (13). car1 car3 double gene disruptants do not aggregate, and developmental gene expression cannot be restored with cAMP, indicating that cAR1 or cAR3 shows functional redundancy and that either one or the other has to be present for gene induction to occur (10, 36). car2 null cells are blocked in the mound stage, while car4 null cells show abnormal slug morphogenesis and culmination. Both lines show reduced expression of prestalk genes and enhanced expression of prespore genes (19, 29).To understand the function of the four cARs, it is essential to know whether each receptor is coupled to a specific signal transduction pathway that controls a specific cell differentiation event or whether each receptor can activate multiple cell differentiation pathways. In the latter case, it is not the presence of a specific receptor that determines whether a response occurs but the availability of the downstream signaling pathway. To determine whether individual receptors have unique functions in developmental gene expression, we examined gene regulation in cell lines that display about equal levels of cAR1, cAR2, and cAR3 in a car1 car3 mutant background. Our results show that with two exceptions, all three receptors can transduce both the excitation and adaptation components of the different cAMP-regulated gene induction events with almost equal levels of efficiency.     

Fatty acid-binding proteins in the heart

Long-chain fatty acids are important fuel molecules for the heart, their oxidation in mitochondria providing the bulk of energy required for cardiac functioning. The low solubility of fatty acids in aqueous solutions impairs their cellular transport. However, cardiac tissue contains several proteins capable of binding fatty acids non-covalently. These fatty acid-binding proteins (FABPs) are thought to facilitate both cellular uptake and intracellular transport of fatty acids. The majority of fatty acids taken up by the heart seems to pass the sarcolemma through a carrier-mediated translocation mechanism consisting of one or more membrane-associated FABPs. Intracellular transport of fatty acids towards sites of metabolic conversion is most likely accomplished by cytoplasmic FABPs. In this review, the roles of membrane-associated and cytoplasmic FABPs in cardiac fatty acid metabolism under (patho)physiological circumstances are discussed.     

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.