Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).     

Plant intraspecific functional trait variation is related to within‐habitat heterogeneity and genetic diversity in Trifolium montanum L.

Intraspecific trait variation (ITV), based on available genetic diversity, is one of the major means plant populations can respond to environmental variability. The study of functional trait variation and diversity has become popular in ecological research, for example, as a proxy for plant performance influencing fitness. Up to now, it is unclear which aspects of intraspecific functional trait variation (iFD_CV) can be attributed to the environment or genetics under natural conditions. Here, we examined 260 individuals from 13 locations of the rare (semi‐)dry calcareous grassland species Trifolium montanum L. in terms of iFD_CV, within‐habitat heterogeneity, and genetic diversity. The iFD_CV was assessed by measuring functional traits (releasing height, biomass, leaf area, specific leaf area, leaf dry matter content, F_v/F_m, performance index, stomatal pore surface, and stomatal pore area index). Abiotic within‐habitat heterogeneity was derived from altitude, slope exposure, slope, leaf area index, soil depth, and further soil factors. Based on microsatellites, we calculated expected heterozygosity (H_e) because it best‐explained, among other indices, iFD_CV. We performed multiple linear regression models quantifying relationships among iFD_CV, abiotic within‐habitat heterogeneity and genetic diversity, and also between separate functional traits and abiotic within‐habitat heterogeneity or genetic diversity. We found that abiotic within‐habitat heterogeneity influenced iFD_CV twice as strong compared to genetic diversity. Both aspects together explained 77% of variation in iFD_CV ( = .77, F_{2, 10} = 21.66, p < .001). The majority of functional traits (releasing height, biomass, specific leaf area, leaf dry matter content, F_v/F_m, and performance index) were related to abiotic habitat conditions indicating responses to environmental heterogeneity. In contrast, only morphology‐related functional traits (releasing height, biomass, and leaf area) were related to genetics. Our results suggest that both within‐habitat heterogeneity and genetic diversity affect iFD_CV and are thus crucial to consider when aiming to understand or predict changes of plant species performance under changing environmental conditions.     

Dynamics of lipid-protein interactions. Interaction of apolipoprotein A-II from human plasma high density lipoproteins with dimyristoylphosphatidylcholine

ApoA-II and dimyristoylphosphatidylcholine (DMPC) spontaneously associate to give three different complexes whose structures are determined by the initial reactant concentration and by the reaction temperature with respect to Tc (23.9 degrees C), the gel to liquid crystalline transition temperature of DMPC. At an initial lipid to protein ratio of 45/1, a single complex (2.29 x 10(5) daltons) is quantitatively formed at all temperatures between Tc - 4 degrees C and Tc + 6 degrees C. When the 45/1 complex is mixed with DMPC liposomes there is lipid exchange but no net transfer of lipid, so that the structure of the complex remains unaltered. At an initial molar ratio of 100 to 300:1, the reaction scheme is more complex. At 24 degrees C a 240/1 complex (1.5 x 10(6) daltons) is formed from a precursor 75/1 complex (3.43 x 10(5) daltons) if excess (approximately 300 mol/mol) lipid is present. The 75/1 complex exhibits lipid exchange in the presence of added DMPC liposomes at 24 degrees C, and both the 75/1 and the 240/1 complex can be converted to smaller protein-rich complexes in the presence of added apoA-II. These results suggest that the initial lipid/protein ratio and the physical state of a lipid or lipid . protein complex determines the composition and structure of the resulting complex and support the view that lipid-protein interactions are stronger than protein-protein or lipid-lipid interactions.     

Frequency of large granular lymphocytes in peripheral blood of healthy persons and breast cancer patients

Summary The frequency of large granular lymphocytes (LGL) in the peripheral blood of healthy persons (n=56) and breast cancer patients (31 cases with stage-I and -II disease and 42 cases with stage-III and -IV disease) was studied. The frequency of LGL in peripheral blood was significantly depressed in cancer patients, and particularly in patients with advanced breast cancer.     

Complementary Roles of the Hippocampus and the Dorsomedial Striatum during Spatial and Sequence-Based Navigation Behavior

We investigated the neural bases of navigation based on spatial or sequential egocentric representation during the completion of the starmaze, a complex goal-directed navigation task. In this maze, mice had to swim along a path composed of three choice points to find a hidden platform. As reported previously, this task can be solved by using two hippocampal-dependent strategies encoded in parallel i) the allocentric strategy requiring encoding of the contextual information, and ii) the sequential egocentric strategy requiring temporal encoding of a sequence of successive body movements associated to specific choice points. Mice were trained during one day and tested the following day in a single probe trial to reveal which of the two strategies was spontaneously preferred by each animal. Imaging of the activity-dependent gene c-fos revealed that both strategies are supported by an overlapping network involving the dorsal hippocampus, the dorsomedial striatum (DMS) and the medial prefrontal cortex. A significant higher activation of the ventral CA1 subregion was observed when mice used the sequential egocentric strategy. To investigate the potential different roles of the dorsal hippocampus and the DMS in both types of navigation, we performed region-specific excitotoxic lesions of each of these two structures. Dorsal hippocampus lesioned mice were unable to optimally learn the sequence but improved their performances by developing a serial strategy instead. DMS lesioned mice were severely impaired, failing to learn the task. Our data support the view that the hippocampus organizes information into a spatio-temporal representation, which can then be used by the DMS to perform goal-directed navigation.     

Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin.

The synthesis of a 5'-O-BzH-2'-O-ACE-protected-3-methyluridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine. Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S ribosomal RNA were generated. Modifications were present at positions 1911, 1915, and 1917. The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy     

The reaction of arsenite-complexed xanthine oxidase with oxygen. Evidence for an oxygen-reactive molybdenum center

The effects of arsenite on the reaction of reduced xanthine oxidase with oxygen are determined. The kinetics of the reaction monitoring the return of enzyme absorbance are investigated as are the kinetics and stoichiometries of peroxide and superoxide formation. Although some of the effects of arsenite are qualitatively consistent with expectations based on the known perturbation of the molybdenum midpoint potentials by arsenite, several results cannot be so easily explained. Specifically, arsenite introduces a very rapid phase (kobs = 110 s-1 at 125 microM oxygen) to the oxidative half-reaction which is not observed with the native enzyme. Arsenite also diminishes the amount of superoxide produced and eliminates one-electron reduced enzyme as a detectable kinetic intermediate in the reoxidation pathway. These differences appear to result from the ability of arsenite to greatly enhance the oxygen- and/or superoxide-reactivity of the reduced molybdenum center. This is reflected in the observation that reduced forms of arsenite-complexed xanthine oxidase lacking functional FAD (iodoacetamide-alkylated enzyme and deflavo enzyme) react relatively rapidly with oxygen whereas these reactions are quite slow in the absence of arsenite.     

The karyotype of Alligator mississippiensis,and chromosomal mapping of the ZFY/X homologue,Zfc

Comparative mapping studies of X-linked genes in mammals have provided insights into the evolution of the X chromosome. Many reptiles including the American alligator, Alligator mississippiensis, do not appear to possess heteromorphic sex chromosomes, and sex is determined by the incubation temperature of the egg during embryonic development. Mapping of homologues of mammalian X-linked genes in reptiles could lead to a greater understanding of the evolution of vertebrate sex chromosomes. One of the genes used in the mammalian mapping studies was ZFX, an X-linked copy of the human ZFY gene which was originally isolated as a candidate for the mammalian testis-determining factor (TDF). ZFX is X-linked in eutherians, but maps to two autosomal locations in marsupials and monotremes, close to other genes associated with the eutherian X. The alligator homologue of the ZFY/ZFX genes, Zfc, has been isolated and described previously. A detailed karyotype of A. mississippiensis is presented, together with chromosomal in situ hybridisation data localising the Zfc gene to chromosome 3. Further chromosomal mapping studies using eutherian X-linked genes may reveal conserved chromosomal regions in the alligator that have become part of the eutherian X chromosome during evolution.