Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox).

The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91(phox) and p22(phox) subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91(phox) are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91(phox) harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91(phox) H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91(phox) H119L or H210L. In contrast, gp65 was not processed to gp91(phox), heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91(phox) H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O(2)-generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His(101), His(115), His(209), and His(222) of gp91(phox) are critical for heme binding and biosynthetic maturation of flavocytochrome b.     

Transport and metabolism of exogenously applied gibberellins to Malus domestica Borkh. cv. Jonagold

The radio-labeled gibberellins GA₁, GA₃,GA₄, and GA₇ were applied to intact developing applefruits (Malus domestica Borkh. cv. Jonagold) during theperiod when GAs are suggested to inhibit flower bud induction for the followingyear. Radioactivity from these compounds was found to be transported intoadjacent tissues as there are pedicels and bourses (4%). Application topedicels, after removal of the fruits, enhanced the transport into adjacentbourses up to 11%. The bud-carrying lateral bourse shoots contained onlyminor amounts of radioactivity on average 0.4% in both cases. Theseexport rates were identical, 1 or 5 days after application.After application of the corresponding deuterium-labeled GAs and analyses bymass spectrometry the specific metabolization of GA₁ toGA₁ 13-O-glucoside and of GA₃ to GA₃13-O-glucoside was demonstrated. Additional metabolites of GA₁ andGA₃ were not detected. After fruit application of GA₃ theratio of GA₃ to GA₃ 13-O-glucoside was found to be 1:2 inthe fruit. Pedicel application led to ratios of 1:4 and 1:5, respectively, inthe pedicel and in the adjacent bourse. After the application of GA₄and GA₇, neither glucosylation products nor other GA-like metabolitescould be identified.This is the first report of the metabolism of GAs to GA 13-O-glucosides indeveloping apple fruits. The possible function of the GAs as a signal in flowerbud formation for the following year is discussed.     

Multifunctional Centromere Binding Factor 1 Is Essential for Chromosome Segregation in the Human Pathogenic Yeast Candida glabrata

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.     

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.     

A biological spectral weighting function for ozone depletion research with higher plants

Biological spectral weighting functions (BSWF) play a key role in assessing implications of stratospheric ozone reduction. They are used to calculate the increase in biologically effective solar UV radiation due to ozone reduction (radiation amplification factor, RAF), assess current latitudinal gradients of solar UV radiation, and compare solar UV radiation with that from lamps and filters used in experiments. As a basis for a BSWF, we developed an action spectrum for growth responses of light-grown oat ( Avena sativa L. cv. Otana) seedlings exposed to narrowband UV radiation from a large double water-prism monochromator. Five UV wavelength peaks were used (275, 297, 302, 313 and 366 nm) in the absence of any visible radiation. Growth responses were measured from 1 to 10 days after the treatments. At all these wavelengths, the UV radiation inhibited height growth, including the height at which the first leaf separated from the stem. Radiation at all wavelengths used, except the one UV-A wavelength, promoted the length of the second leaf. The resulting action spectrum closely resembles the commonly used generalized plant response function except that it indicates continued sensitivity into the UV-A region. When used as a BSWF for the ozone depletion problem, this new function for plant growth would suggest substantially less impact of ozone depletion because it results in only a modest increment of biologically effective UV for a given level of ozone depletion (a lower RAF). Yet this new BSWF also suggests that experimental treatments based on previous BSWF with less emphasis on the UV-A may have resulted in simulations of less pronounced ozone depletion than intended. The validity of this new BSWF with UV-A sensitivity, designated the UV plant growth weighting function, was verified in field experiments as described in the companion paper.     

St Lucia Whiptail Lizard Cnemidophorus vanzoi (Sauria: Teiidae) microsatellite primers

Eight polymorphic microsatellite loci have been characterized for the endangered St Lucia Whiptail Lizard Cnemidophorus vanzoi. Endemic to two small islets, Maria Major and Maria Minor, off the coast of St Lucia, Lesser Antilles, the world population is estimated at < 1000 individuals. However, representatives of the systematically complex genus Cnemidophorus, containing sexual species and parthenogenetic allopolyploids, are distributed widely in North, Central and South America, and on several islands in the Caribbean Sea. These microsatellite markers are being used to monitor the genetic structure of a population of St Lucia Whiptails, recently founded through translocation, on the nearby Praslin Island.     

Vision and Eating Behavior

Objective: Eating behavior is influenced by internal and external factors. Vision is one part of the complex pattern of factors influencing the amount of food consumed during a meal. The aim of this study was to explore the impact of vision on the microstructure of eating behavior and the subjective motivation to eat. Research Methods and Procedures: Nine blind subjects and nine matched seeing control subjects consumed a standardized meal registered by VIKTOR, an eating monitor, measuring the microstructure of the eating behavior. The eating behavior of the control subjects was registered twice, with and without blindfold. Results: The eating behavior of the blind subjects did not differ from that of seeing control subjects. However, the eating behavior of seeing subjects eating with blindfold demonstrated a clear impact of vision on eating behavior. When blindfolded, subjects ate 22% less food (p < 0.05), had shorter meal durations (p < 0.05), and had less decelerated eating curves (p < 0.05). Despite a smaller amount of food consumed when blindfolded, the reported feeling of fullness was identical to that reported after the larger meal consumed without blindfold. Discussion: The importance of vision in regulating our eating behavior is further stressed in this study. Eating with a blindfold decreased the intake of food, without making subjects feel less full. Eating blindfolded, therefore, may force subjects to rely more on internal signals. These results might be used as an aid in the development of new treatment strategies for obese subjects.     

Deficiency of SP-B reveals protective role of SP-C during oxygen lung injury.

Although the surface properties of surfactant protein (SP)-B and SP-C are similar, the contributions that either protein may make to lung function have not been identified in vivo. Mutations in SP-B cause lethal respiratory failure at birth; however, SP-B null mice are deficient in both SP-B and SP-C. To identify potential contributions of SP-C to lung function in vivo, the following transgenic mice were generated and exposed to 95% O(2) for 3 days: (SP-B(+/+),SP-C(+/+)), (SP-B(+/+), SP-C(-/-)), (SP-B(+/-),SP-C(+/+)), (SP-B(+/-),SP-C(+/-)), and (SP-B(+/-),SP-C(-/-)). Hyperoxia altered pressure-volume curves in mice that were heterozygous for SP-B, and these values were further decreased in (SP-B(+/-),SP-C(-/-)) mice. Likewise, alveolar interleukin (IL)-6 and IL-1 beta were maximally increased by O(2) exposure of (SP-B(+/-),SP-C(-/-)) mice compared with the other genotypes. Lung hysteresivity was lower in the (SP-B(+/-),SP-C(-/-)) mice. Surfactant isolated from (SP-B(+/+),SP-C(-/-)) and (SP-B(+/-),SP-C(-/-)) mice failed to stabilize the surface tension of microbubbles, showing that SP-C plays a role in stabilization or recruitment of phospholipid films at low bubble radius. Genetically decreased levels of SP-B combined with superimposed O(2)-induced injury reveals the distinct contribution of SP-C to pulmonary function in vivo.     

Quantitative relationship between indole-3-acetic acid and abscisic acid during leaf growth in Coleus blumei

Quantitative determinations by gas chromatography-mass spectrometry ofindole-3-acetic acid (IAA) and abscisic acid (ABA) in growing leaves ofColeusblumei plants show parallel declines in leaf concentrations of bothhormones,except in leaf number 3 (about three-fourths of full size) where IAA level wasthe lowest of those measured. Expansion of the most recently unfurled leaf tofull size serves, in effect, to dilute both IAA and ABA about 1.7 to 1.Althoughabsolute levels of leaf IAA varied as much as an order of magnitude from onebatch of plants to another, ABA levels were proportional to the IAA level withan overall correlation coefficient of 0.91. Evidence, both correlative andcausal, for the determination of ABA status by IAA—and of IAA status byABA—in leaves and other developing organs is summarized.