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Cells respond to environmental cues by modifying protein complexes in the nucleus to produce a change in the pattern of gene expression. In this article, we review techniques that allow us to visualize these protein interactions as they occur in living cells. The cloning of genes from marine organisms that encode fluorescent proteins provides a way to tag and monitor the intracellular behavior of expressed fusion proteins. The genetic engineering of jellyfish green fluorescent protein (GFP) and the recent cloning of a sea anemone red fluorescent protein (RFP) have provided fluorescent tags that emit light at wavelengths ranging from the blue to the red spectrum. Several of these color variants can be readily distinguished by fluorescence microscopy, allowing them to be used in combination to monitor the behavior of two or more independent proteins in the same living cell. We describe the use of this approach to examine where transcription factors are assembled in the nucleus. To demonstrate that these labeled nuclear proteins are interacting, however, requires spatial resolution that exceeds the optical limit of the light microscope. This degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). The application of FRET microscopy to detect the interactions between proteins labeled with the color variants of GFP and the limitations of the FRET approach are discussed. The use of different-color fluorescent proteins in combination with FRET offers the opportunity to study the complex behavior of key regulatory proteins in their natural environment within the living cell.

We evaluated whether fish oil or vitamin E administration affected ethanol-induced changes in membrane ATPases. Male Wistar rats (225-250 g) were fed, through a gastric tube a liquid diet containing fish oil (25% of calories) and ethanol for one month. Another group of animals was given supplemental vitamin E (300 u/kg). In the pair-fed control animals, ethanol-derived calories were replaced with dextrose. The blood ethanol levels were maintained between 150 and 350 mg/dL. At sacrifice, the red cells were immediately washed with ice-cold saline, membranes were prepared and ATPases measured. These was no difference in the Na+K+ ATPase, Ca2+ ATPase and Mg2+ ATPase activities between the fish oil-dextrose and corn oil-dextrose groups. A decrease in Ca2+ ATPase and an increase in Na+K+ ATPase was seen with ethanol feeding; these change are similar to those seen in corn oil-ethanol fed rats. In contrast, Vitamin E administration prevented the ethanol-induced changes in ATPase. This observation provides support for the role of lipid peroxidation in alcohol-induced changes in cell membrane ATPase activities.

Acetic acid bacteria such as Gluconobacter oxydans are used in several biotechnological processes due to their ability to perform rapid incomplete regio- and stereo-selective oxidations of a great variety of carbohydrates, alcohols, and related compounds by their membrane-bound dehydrogenases. In order to understand the growth physiology of industrial strains such as G. oxydans ATCC 621H that has high substrate oxidation rates but poor growth yields, we compared its genome sequence to the genome sequence of strain DSM 3504 that reaches an almost three times higher optical density. Although the genome sequences are very similar, DSM 3504 has additional copies of genes that are absent from ATCC 621H. Most importantly, strain DSM 3504 contains an additional type II NADH dehydrogenase (ndh) gene and an additional triosephosphate isomerase (tpi) gene. We deleted these additional paralogs from DSM 3504, overexpressed NADH dehydrogenase in ATCC 621H, and monitored biomass and the concentration of the representative cell components as well as O2 and CO2 transfer rates in growth experiments on mannitol. The data revealed a clear competition of membrane-bound dehydrogenases and NADH dehydrogenase for channeling electrons in the electron transport chain of Gluconobacter and an important role of the additional NADH dehydrogenase for increased growth yields. The less active the NADH dehydrogenase is, the more active is the membrane-bound polyol dehydrogenase. These results were confirmed by introducing additional ndh genes via plasmid pAJ78 in strain ATCC 621H, which leads to a marked increase of the growth rate.

Double Environmental Injustice The escalating effects of hurricanes on population health represent a double environmental injustice: disadvantaged populations sustain disproportionate harm, and tho...

Abstract— The lowest excited singlet and triplet states of coumarin, psoralen, and 4‐hydroxy‐coumarin have been assigned to the (π,π*) type on the basis of the luminescence spectroscopy and MO calculations. The mechanism of photocycloaddition of courmarin and psoralen to thymine has been described in terms of the perturbational MO model and MO reactivity indices. All possible cycloaddition patterns have been examined. Results suggest that the 3,4‐bond of coumarin in the excited state is somewhat more reactive than the same bond of psoralen in the excited state. It is also predicted that the 3,4‐bond of psoralen in the triplet state is more reactive than the 4′, 5′‐bond. The results have been favorably correlated with the electronic characteristics of excited coumarin molecules and with available experimental data on the relative yields of photoadducts.

In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.

ABSTRACTThe aquifer system of Doñana (SW Spain) represents the most important freshwater source in the Doñana Natural Area. Its spatiotemporal dynamics favours the hydrological connection between surface and subsurface ecosystems, and promotes matter fluxes among the different terrestrial and aquatic systems present here. This aquifer has been intensively studied from a hydrogeological point of view but little is known from an ecological perspective. In order to understand the ecological roles played by microbial communities in this system, we conducted a long‐term seasonal study of bacterial abundance, cell biomass, bacterial biomass and functional activities over a 2‐year period. Bacterial abundance ranged between 2.11 ± 1.79 × 105 and 8.58 ± 6.99 × 107 bacteria mL−1 groundwater, average cell biomass was estimated to be 77.01 ± 31.56 fgC and bacterial biomass varied between 8.99 ± 4.10 × 10−2 and 5.65 ± 0.70 µgC mL−1. Iron‐related bacteria showed the highest activities among the functional groups studied. Moreover, among the variables that usually control spatial distributions of microbial communities in aquifer systems, depth did not have a relevant effect on this aquifer, at least in the range of depths studied, but grain size, probably due to its direct effects on hydrogeological parameters, such as permeability or porosity, appeared to exert moderate control, principally in terms of bacterial abundance. Finally, significant seasonal differences in the means of these microbiological variables were also observed; temperature seems to be the main factor controlling the temporal distribution of microbial communities in this aquifer system.

The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S.cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations: a collection of collapsed structures with an average fluorophore-heme distance (r) of 27 A and a roughly equal population of extended polypeptides with r > 50 A. Molecules with the native fold appear on a time scale regulated by heme ligation events ( approximately 300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure.

Rostromedial tegmental nucleus (RMTg) GABA neurons exert a primary inhibitory drive onto midbrain dopamine neurons and are excited by a variety of aversive stimuli. There is, however, little evidence that the RMTg-ventral tegmental area (VTA)-nucleus accumbens shell (Acb) circuit plays a role in the aversive consequences of alcohol withdrawal. This study was performed in adult male Long-Evans rats at 48-h withdrawal from chronic alcohol drinking in the intermittent schedule. These rats displayed clear anhedonia and depression-like behaviors, as measured with the sucrose preference, and forced swimming tests. These aberrant behaviors were accompanied by a substantial increase in cFos expression in the VTA-projecting RMTg neurons, identified by a combination of immunohistochemistry and retrograde-tracing techniques. Pharmacological or chemogenetic inhibition of RMTg neurons mitigated the anhedonia and depression-like behaviors. Ex vivo electrophysiological data showed that chemogenetic inactivation of RMTg neurons reduced GABA release and accelerated spontaneous firings of VTA dopamine neurons. Finally, using a functional hemispheric disconnection procedure, we demonstrated that inhibition of unilateral RMTg, when combined with activation of D1 and D2 dopamine receptors in the contralateral (but not ipsilateral) Acb, mitigated the anhedonia and depression-like behaviors in alcohol-withdrawal rats. These data show that the integrity in the RMTg-VTA-Acb pathway in a single hemisphere is sufficient to elicit depression-like behavior during ethanol-withdrawal. Overall, the present results reveal that the RMTg-VTA-Acb pathway plays a crucial role in the depression-like behavior in animals undergoing alcohol withdrawal, further advocating the RMTg as a potential therapeutic target for alcoholism.

The effect of carbon source on efficacy of anaerobic soil disinfestation (ASD) toward suppression of apple root infection by Rhizoctonia solani AG-5 and Pratylenchus penetrans was examined. Orchard grass (GR), rice bran (RB), ethanol (ET), composted steer manure (CM), and Brassica juncea seed meal (SM) were used as ASD carbon inputs, with plant assays conducted in natural and pasteurized orchard soils. Subsequent studies investigated the effect of GR application rate used in ASD on control of these pathogens. In general, apple root infection by R. solani AG-5 was significantly lower in ET, GR, RB, and SM ASD treatments compared with the control. Among different ASD treatments, apple seedling growth was significantly greater when GR or SM was used as the carbon input relative to all other ASD treatments. R. solani AG-5 DNA abundance was significantly reduced in all ASD treatments, regardless of amendment type, compared with the control. In independent experiments, ASD-GR was consistently superior to ASD-CM for limiting pathogen activity in soils. ASD treatment with a grass input rate of 20 t ha−1provided superior suppression of P. penetrans but grass application rate did not affect ASD efficacy in control of R. solani AG-5. The soil microbiome from ASD-GR-treated soils was clearly distinct from the control and ASD-CM-treated soils. In contrast, composition of the microbiome from control and ASD-CM-treated soils could not be differentiated. Comparative results from pasteurized and nonpasteurized soils suggest that there is potential for GR based ASD treatment to recruit microbial elements that persist over the anaerobic phase of soil incubation, which may functionally contribute to disease suppression. When ASD was conducted with GR, microbial diversity was markedly reduced relative to the control or ASD-CM soil suggesting that this parameter, typically associated with system resilience, was not instrumental to the function of ASD-induced soil suppressiveness.