Evidence for a specific microwave radiation effect on the green fluorescent protein

We have compared the effect of microwave irradiation and of conventional heating on the fluorescence of solution-based green fluorescent protein. A specialized near-field 8.5 GHz microwave applicator operating at 250 mW input microwave power was used. The solution temperature, the intensity, and the spectrum of the green fluorescent protein fluorescence 1), under microwave irradiation and 2), under conventional heating, were measured. In both cases the fluorescence intensity decreases and the spectrum becomes red-shifted. Although the microwave irradiation heats the solution, the microwave-induced changes in fluorescence cannot be explained by heating alone. Several possible scenarios are discussed.     

Universal stress proteins are important for oxidative and acid stress resistance and growth of Listeria monocytogenes EGD-e in vitro and in vivo

Background

Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species.

Methods and Findings

We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (Δlmo0515, Δlmo1580 and Δlmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models.Tolerance to acidic stress was clearly reduced in Δlmo1580 and Δlmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in Δlmo1580 and Δlmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with Δlmo1580 or Δlmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection.

Conclusion

This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions.     

Dynamic Interactions between Root NH4+ Influx and Long-Distance N Translocation in Rice: Insights into Feedback Processes

Ammonium influx into roots and N translocation to the shootswere measured in 3-week-old hydroponically grown rice seedlings(Oryza sativa L., cv. IR72) under conditions of N deprivationand NH₄⁺ resupply, using ¹³NH₄⁺as a tracer. Root NH₄⁺ influxwas repressed in plants continuously supplied with NH₄⁺ (at0.1 mM), but a high proportion of absorbed N (20 to 30%) wastranslocated to the shoot in the form of N assimilates duringthe 13-min loading and desorption periods. Interruption of exogenousNH₄⁺ supply for periods of 1 to 3 d caused NH₄⁺ influx to bede-repressed. This same treatment caused N translocation tothe shoot to decline rapidly, until, by 24 h, less than 5% ofthe absorbed ¹³N was translocated to the shoot, illustratinga clear priority of root over shoot N demand under conditionsof N deprivation. Upon resupplying 1 mM NH₄⁺, root NH₄⁺ influxresponded in a distinct four-phase pattern, exhibiting periodsin which NH₄⁺ influx was first enhanced and subsequently reduced.Notably, a 25 to 40% increase in root influx, peaking at {smalltilde}2 h following re-exposure was correlated with a 4- to5-fold enhancement in shoot translocation and a repression ofroot GS activity. The transient increase of NH₄⁺ influx wasalso observed in seedlings continuously supplied with NO₃^–and subsequently transferred to NH₄⁺. Extended exposure to NH₄⁺caused root NH₄⁺ influx to decrease progressively, while shoottranslocation was restored to {small tilde}30% of incoming NH₄⁺.The nature of the feedback control of NH₄⁺ influx as well asthe question of its inducibility are discussed. (Received August 7, 1998; Accepted September 21, 1998)     

Arabidopsis 10-formyl tetrahydrofolate deformylases are essential for photorespiration

In prokaryotes, PurU (10-formyl tetrahydrofolate [THF] deformylase) metabolizes 10-formyl THF to formate and THF for purine and Gly biosyntheses. The Arabidopsis thaliana genome contains two putative purU genes, At4g17360 and At5g47435. Knocking out these genes simultaneously results in plants that are smaller and paler than the wild type. These double knockout (dKO) mutant plants show a 70-fold increase in Gly levels and accumulate elevated levels of 5- and 10-formyl THF. Embryo development in dKO mutants arrests between heart and early bent cotyledon stages. Mature seeds are shriveled, accumulate low amounts of lipids, and fail to germinate. However, the dKO mutant is only conditionally lethal and is rescued by growth under nonphotorespiratory conditions. In addition, culturing dKO siliques in the presence of sucrose restores normal embryo development and seed viability, suggesting that the seed and embryo development phenotypes are a result of a maternal effect. Our findings are consistent with the involvement of At4g17360 and At5g47435 proteins in photorespiration, which is to prevent excessive accumulation of 5-formyl THF, a potent inhibitor of the Gly decarboxylase/Ser hydroxymethyltransferase complex. Supporting this role, deletion of the At2g38660 gene that encodes the bifunctional 5,10-methylene THF dehydrogenase/5,10-methenyl THF cyclohydrolase that acts upstream of 5-formyl THF formation restored the wild-type phenotype in dKO plants.     

A Protease Activity Displaying Some Thrombin-like Characteristics in Conditioned Medium of Zinnia Mesophyll Cells Undergoing Tracheary Element Differentiation

The normal development of tracheary elements (TE) requires a selective degradation of the cytoplasm without loss of the extracellular wall that remains behind as the water-conducting units of xylem. Using zinnia-(Zinnia elegans L. cv. Green Envy) cultured mesophyll cells that synchronously transdifferentiate into TEs, extracellular and intracellular proteases, respectively, have been shown to both trigger death and to execute autolysis as the final component of a programmed cell death (PCD). We report here the appearance in the medium of an unusual proteolytic activity correlated with the PCD process just prior to the autolysis. The activity has a pH optimum of 5.5–6.0 and displays some thrombin characteristics. This protease activity has 1) a 10-fold higher affinity towards a thrombin-specific chromogenic substrate than toward a trypsin-specific chromogenic substrate; 2) a 1000-fold lower sensitivity to soybean trypsin inhibitor (STI) compared to trypsin; and 3) limited ability to cleave the protease-activated receptor-1, the native thrombin substrate. However, the addition of partially purified fraction containing the thrombin-like protease activity to the medium of PCD-competent cells does not prematurely trigger PCD, and the thrombin-specific peptide inhibitor phenylalanine-proline-aspartic acid-chloromethylketone fails to inhibit PCD or tracheary element (TE) formation. This suggests that this protease activity may play a role within the cells in execution of the autolysis or in the collapse of the tonoplast rather than as an extracellular proteolytic activity participating in the chain of events leading to cell death. Online publication: 7 April 2005     

Functional polymorphism in lycopene beta-cyclase gene as a molecular marker to predict bixin production in <Emphasis Type="Italic">Bixa orellana</Emphasis> L. (achiote)

Bixin is an apocarotenoid obtained from the seed aril of Bixa orellana L., a tropical plant known as achiote in Mexico. This compound is the second most commonly used natural colouring for food and pharmaceutical industries. B. orellana is an outcrossing species that displays high genetic variability. Recently, the colour traits of sexual organs were associated with the biosynthesis and accumulation of bixin in mature seeds. Herein, we describe a new approach for genotype–phenotype association by surveying lycopene beta-cyclase (Boβ-LCY1) gene variation in sixteen achiote accessions divided into three groups according to contrasting traits, such as flower colour, fruit colour and bixin production. Using a combination of single-strand conformational polymorphism techniques and the sequencing of polymorphic bands, we identified several single-nucleotide polymorphisms that divided the accessions into three haplotypes. Surprisingly, we observed that these three haplotypes were consistent with the same three groups previously characterized by phenotypic traits. We derived a putative sequence for the Boβ-LCY1 gene and surveyed the variations in this sequence. The heterozygosity of Boβ-LCY1 alleles resulted in a higher bixin content, likely associated with heterosis for this metabolite. These findings augment the toolbox available for the selection and genetic improvement of B. orellana and provide a reliable phenotype–genotype association method for commercial varietal selection, contributing to the development of laboratory techniques to identify desirable traits of commercial plant species.     

Functional coupling between HIV-1 integrase and the SWI/SNF chromatin remodeling complex for efficient in vitro integration into stable nucleosomes

Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier.