Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)

Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.     

Separating the climatic signal from tree-ring width and maximum latewood density records

We propose a technique for separating the climatic signal which is contained in two tree-ring parameters widely used in dendroclimatology. The method is based on the removal of the relationship between tree-ring width (TRW) and maximum latewood density (MXD) observed for narrow tree rings from high latitudes. The new technique is tested on data from three larch stands located along the northern timberline in Eurasia. Correlations were calculated between the temperatures of pentads (five consecutive days), TRW chronologies and MXD chronologies calculated according to the standard and proposed methods. The analysis confirms the great importance of summer temperature for tree radial growth and tree-ring formation. TRW is positively correlated with the temperature of four to eight pentads (depending on the region) at the beginning of the growth season, but MXD as obtained by the standard technique is correlated with temperature over a much longer period. For maximum density series from which the relationship between MXD and TRW has been removed (MXD′), there is a clear correlation with temperatures in the second part of the growing season. These results are consistent with the known dynamics of tree-ring growth in high latitudes and mechanisms of tree-ring formation.     

Functional asymmetry of the F0 motor in bacterial ATP synthases

F₁F₀ ATP synthases use the electrochemical potential of H⁺ or Na⁺ across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F₀ parts of a Na⁺- and H⁺-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of ∼100 times higher Na⁺ or H⁺ concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F₀ parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo . In Escherichia coli , we observed respiratory chain-driven ATP production at pH 7–8, while P -site pH values < 6.5 were required for ATP synthesis in vitro . This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.     

The effects of diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-SECO-19-norpregna-4,5-diene-3,10,20-trione (SECO) on androgen biosynthesis in the rat testis and epididymis

We have investigated the effects of two 4-ene-steroid 5 alpha-reductase inhibitors, diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and (4R)-5,10-seco-19-norpregna-4, 5-diene-3,10,20-trione (SECO), on testicular and epididymal androgen biosynthesis. Kinetic analyses revealed that both compounds inhibited epididymal DHT biosynthesis. 4-MA was a competitive inhibitor of epididymal nuclear and microsomal 4-ene-steroid 5 alpha-reductases (3-oxo-5 alpha-steroid: NADP 4-ene-oxidoreductase EC 1.3.1.22) with Kiapp values of 12.8 and 15.1 nmol/l compared to the respective Kmapp values of 185 and 240 nmol/l. Values for the Vmaxapp were always within 70-130% of the control. SECO at 1.0 mumol/l, also inhibited epididymal nuclear and microsomal 4-ene-steroid-5 alpha-reductases, causing respectively 2.9 and 5.2-fold increases in Kmapp. The Vmaxapp values were unchanged. However, SECO concentrations of 5 and 25 mumol/l abolished 4-ene-steroid 5 alpha-reductase activity at all testosterone concentrations. To examine the specificity of these compounds, we investigated their effects on the enzymes that convert pregnenolone to testosterone. Rat testis microsomes converted pregnenolone to testosterone via the 4-ene-3-oxo pathway, with the major metabolites being progesterone, 17-hydroxyprogesterone, 4-androstenedione and testosterone; some 17-hydroxypregnenolone was also formed. Very small amounts of dehydroepiandrosterone (DHA) and 5-androstenediol were detected. SECO, at a concentration that completely inhibited epididymal 4-ene-steroid 5 alpha-reductase activity, did not alter the metabolic profile of pregnenolone metabolism. However, 4-MA prevented the appearance of 4-ene steroids, and large quantities of 17-hydroxypregnenolone and DHA accumulated, suggesting that inhibition of the 3 beta-hydroxysteroid: NAD(P)+ oxidoreductase (EC 1.1.1.51) and 3-oxosteroid 5-ene-4-ene-isomerase (EC 5.3.3.1) [3 beta-hydroxysteroid dehydrogenase-isomerase] was occurring. Optimal conditions for the microsomal conversion of DHA to 4-androstenedione were determined; kinetic analyses of the 3 beta-hydroxysteroid dehydrogenase-isomerase activity revealed that 4-MA inhibited this reaction non-competitively, reducing Vmaxapp values to 25% of the control. The Kiapp determined from the intercept replot, was 121 nmol/l, and the Kmapp was always between 90 and 130% of the control value. It is concluded that SECO is more specific than 4-MA in its effects on androgen biosynthesis in the testis and epididymis and that both these drugs should provide useful tools in assessments of the relative contributions of 5 alpha-reduced androgens to androgen dependent processes.     

Proteins of the inner and outer centromere of mitotic chromosomes

We have used immunocytochemistry and molecular cloning methods to identify and characterize structural polypeptides of the centromere. These studies permit us to resolve two distinct regions: the inner and outer centromere. (i) Components of the outer centromere: autoantibodies from certain patients with rheumatic disease identify a family of three immunologically related polypeptides that we have designated CENP-A (17 kDa), CENP-B (80 kDa), and CENP-C (140 kDa). CENP-B has been cloned and sequenced. DNA sequence analysis indicates that this polypeptide possesses two large regions with extraordinary concentrations of acidic residues (region I: 61 residues with 79% glu + asp; region II: 31 residues with 87% glu + asp). Despite this concentration of negative charge, immunocytochemical experiments suggest that CENP-B may be a DNA binding protein. In these experiments, the levels of CENP-B are seen to vary reproducibly from chromosome to chromosome. The role of CENP-B in vivo is unknown. However, it is unlikely to bind directly to the spindle microtubules since it is found at an inactive centromere that apparently does not attach to the spindle. (ii) Components of the inner centromere: we have injected mice with the whole chromosome scaffold fraction to elicit production of monoclonal antibodies. One such antibody identifies two structurally related polypeptides (the INCENP antigens, 135 and 155 kDa) that are preferentially located between the sister chromatids at the centromere. The INCENP antigens undergo dramatic movements from the chromosomes to the central spindle during mitosis. They are ultimately sequestered in the midbody and discarded. Several lines of evidence suggest that the INCENP polypeptides may be involved in the regulation of sister chromatid separation at the metaphase-anaphase transition.     

Survival of Escherichia coli in the environment: fundamental and public health aspects

In this review, our current understanding of the species Escherichia coli and its persistence in the open environment is examined. E. coli consists of six different subgroups, which are separable by genomic analyses. Strains within each subgroup occupy various ecological niches, and can be broadly characterized by either commensalistic or different pathogenic behaviour. In relevant cases, genomic islands can be pinpointed that underpin the behaviour. Thus, genomic islands of, on the one hand, broad environmental significance, and, on the other hand, virulence, are highlighted in the context of E. coli survival in its niches. A focus is further placed on experimental studies on the survival of the different types of E. coli in soil, manure and water. Overall, the data suggest that E. coli can persist, for varying periods of time, in such terrestrial and aquatic habitats. In particular, the considerable persistence of the pathogenic E. coli O157:H7 is of importance, as its acid tolerance may be expected to confer a fitness asset in the more acidic environments. In this context, the extent to which E. coli interacts with its human/animal host and the organism''s survivability in natural environments are compared. In addition, the effect of the diversity and community structure of the indigenous microbiota on the fate of invading E. coli populations in the open environment is discussed. Such a relationship is of importance to our knowledge of both public and environmental health.