Extended Water-Level Drawdowns in Dammed Rivers Enhance Fish Habitat: Environmental Pool Management in the Upper Mississippi River.

Environmental Pool Management (EPM) can improve ecosystem function in rivers by restoring aspects of the natural flow regime lost to dam construction. EPM recreates summer baseflow conditions and promotes the growth of terrestrial vegetation which is inundated in the fall, thereby improving habitat heterogeneity for many aquatic taxa. A three-year experiment was conducted wherein terrestrial floodplain areas were dewatered through EPM water-level reductions and the resulting terrestrial vegetation was (1) allowed to remain or (2) removed in paired plots in Mississippi River pool 25. Fish assemblage and abundance were quantified in paired plots after inundation. Abundances of many fish species were greater in vegetated plots, especially for species that utilize vegetation during portions of their life history. Fish assemblages varied more between plot types when the magnitude of EPM water-level drawdowns was greater, which produced greater vegetation growth. Young-of-year individuals, especially from small, early maturing species and/or species reliant on vegetation for refuge, feeding, or life history, utilized vegetated plots more than devegetated plots. Vegetation growth produced under EPM was heavily used by river fishes, including young-of-year individuals, which may ultimately positively influence recruitment. Increased habitat heterogeneity may mitigate some of the negative impacts of dam construction and water-level regulation on river fishes. Annual variability in vegetation responses that occurs under EPM enhances natural environmental variability which could ultimately contribute to increased fish diversity. Low-cost programs like EPM can be implemented as a part of adaptive management plans to help maintain biodiversity and ecosystem health in anthropogenically altered rivers.

Population demographics of American eels Anguilla rostrata in two Arkansas, U.S.A., catchments that drain into the Gulf of Mexico.

The goal of this study was to compare American eel Anguilla rostrata life history in two inland river systems in Arkansas, U.S.A., that ultimately discharge into the Gulf of Mexico via the Mississippi River and the Red-Atchafalaya catchments. From 21 June 2011 to 24 April 2014, 238 yellow-phase A. rostrata were captured in the middle Ouachita River and tributaries using boat electrofishing and 39 in the lower White River using multiple sampling gears. Most of them were caught downstream of dams in both basins (61%). Medium-sized A. rostrata ranging from 225 to 350 mm total length (LT ) were the most abundant size group in the Ouachita River basin, but they were absent from the White River. Mean LT at age 4 years (i.e. youngest shared age) was 150 mm greater for the White River than the Ouachita River basin. Anguilla rostrata appeared to have a greater initial LT (i.e. minimum size upon arrival) in the White River that allowed them to reach a gonado-somatic index (IG ) of 1·5 up to 4 years earlier, and downstream migration appeared to occur 5 years earlier at 100 mm greater LT ; these differences may be related to increased river fragmentation by dams in the Ouachita River basin. Growth and maturation of A. rostrata in this study were more similar to southern populations along the Atlantic coast than other inland populations. Adult swimbladder nematodes Anguillicoloides crassus were not present in any of the 214 swimbladders inspected. Gulf of Mexico catchments may be valuable production areas for A. rostrata and data from these systems should be considered as range-wide protection and management plans are being developed.

Cytochrome c is released in a single step during apoptosis.

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).

Using flowering times and leaf numbers to model the phases of photoperiod sensitivity in Antirrhinum majus L.

A model has been developed that can be used to determine the phases of sensitivity to photoperiod for seedlings subjected to reciprocal transfers at regular intervals between long (LD) and short day (SD) conditions. The novel feature of this approach is that it enables the simultaneous analysis of the time to flower and number of leaves below the inflorescence. A range of antirrhinum cultivars were grown, all of which were shown to be quantitative long-day plants. Seedlings were effectively insensitive to photoperiod when very young (juvenile). However, after the end of the juvenile phase, SD delayed flowering and increased the number of leaves below the inflorescence. Plants transferred from LD to SD showed a sudden hastening of flowering and a decrease in leaf number once sufficient LD had been received for flower commitment. Photoperiod had little effect on the rate of flower development. The analysis clearly identified major cultivar differences in the length of the juvenile phase and the photoperiod-sensitive inductive phase in both LD and SD.

Partial purification of tomato fruit peroxidase and its effect on the mechanical properties of tomato fruit skin.

Peroxidase (EC 1.11.1.7)-mediated stiffening of cell walls within the fruit skin of tomato is hypothesized to regulate fruit growth. However, to date, there is no experimental evidence demonstrating that peroxidase affects the mechanical properties of skin tissue. Here, the mechanical properties of skin strips excised from a range of fruits at different ages were determined using an 'Instron' universal material testing instrument. The stiffness of tomato fruit skin strips increases 3-fold with increasing fruit age. Application of partially-purified peroxidase from the cell walls of mature tomato fruit skin significantly increased the stiffness of fruit skin irrespective of the age of fruit. Furthermore, the application of hydrogen peroxide significantly increased the stiffness of skin strips excised from fruit of an age when endogenous peroxidase isozymes associated with the termination of growth are first detected. The results support the hypothesis that the tomato fruit skin plays an integral role in the regulation of tomato fruit growth, and that changes in its mechanical properties may be mediated by peroxidase. As far as is known, this is the first demonstration that peroxidases alter the mechanical properties of the plant cell wall.

Subcellular localization of peroxidase in tomato fruit skin and the possible implications for the regulation of fruit growth.

The cessation of tomato fruit growth has been associated with the appearance of three 'wall-bound' peroxidase isozymes in the skin of tomato fruit. However, the presence of these isozymes in the ionically eluted 'wall-bound' fraction may be an artefact of either non-specific binding of symplastic peroxidase to the cell wall, or isozymes bound to membranes included in the 'wall-bound' fraction. Therefore, subcellular localization of peroxidase in both immature and mature tomato fruit skins was studied. Immature fruits showed intense peroxidase activity associated with the tonoplast and pro-vacuolar membranes, but little or no activity associated with the cell wall. However, the presence of peroxidase activity within the cell wall of mature green fruits was confirmed. Furthermore, peroxidase activity was also observed associated with the plasma membrane and large vesicles allied to the plasma membrane. While cross-linking in cell wall components was previously assumed to be the mechanism by which peroxidase might control fruit growth, the incorporation of 'lignin-like' phenolics may also play a part. Isoelectric focusing (IEF) of both symplastic and apoplastic peroxidase extracted from immature and mature tomato fruit skin showed that all peroxidase isozymes present were highly anionic. In this current study, histochemical techniques are used to demonstrate a developmental increase in 'lignin-like' phenolics within the sub-cuticular cell walls of the fruit skin. The localization of peroxidase within tomato fruit skin is discussed in relation to its potential role in the regulation of tomato fruit growth.

Improving quantitative flowering models through a better understanding of the phases of photoperiod sensitivity.

A quantitative understanding of the phases of sensitivity to photo-thermal environment is important if the accuracy of flowering models is to be improved and if the timing of long and short day treatments in protected cropping is to be optimized. A simple method of quantifying the duration of the phases of sensitivity to photoperiod is through the use of reciprocal transfer experiments where plants are transferred between long and short days at regular intervals throughout development. The advantages and disadvantages of different analytical approaches used to analyse such data sets are examined. Inconsistencies between the approaches are highlighted, as are differences in the way authors have interpreted data. The problem of confounding the effects of photoperiod and light integral is considered, as is the need to separate the number of inductive cycles needed for flower commitment from the length of the juvenile phase. The effects of photo-thermal environment on the duration of these phases of photoperiod sensitivity are discussed, together with topics requiring further development.

Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator.

To investigate the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of approximately 2 microM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.

Inhibition of NF-kappa B activation by arsenite through reaction with a critical cysteine in the activation loop of Ikappa B kinase.

Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism by which arsenite affects the NF-kappaB signaling pathway. Activation of transcription factor NF-kappaB depends on the integrity of the IkappaB kinase (IKK) complex. We found that arsenite potently inhibits NF-kappaB and IKK activation by binding to Cys-179 in the activation loop of the IKK catalytic subunits, IKKalpha/beta. The affinity of IKKbeta for trivalent arsenic was verified in vitro by the ability of IKKbeta to enhance the fluorescence of an arsenic-substituted fluorescein dye. The addition of 1,2-dithiol antidotes or replacement of Cys-179 with an alanine residue abolished dye binding to and arsenite inhibition of IKKbeta. Overexpression of IKKbeta (C179A) protects NF-kappaB from inhibition by arsenite, indicating that despite the involvement of a large number of distinct gene products in this activation pathway, the critical target for inhibition by arsenite is on the IKK catalytic subunits.

Understanding women who are violent in intimate relationships: implications for Army family advocacy.

Women who are violent in intimate relationships is a controversial and neglected subject in the area of spouse abuse in the civilian and military communities. Researchers report that women initiate more acts of violence than their male partners. This article provides a review of the literature, which identifies the high rates of violence by women against their male partners. In addition, this article discusses the context in which women offend and the motivations of women offenders. The implication for the Army Family Advocacy Program (FAP) is to enhance providers' clinical knowledge and increase community members' awareness so that FAP personnel can appropriately intervene with abusive couples. The goal of this author is to argue for broadening the scope of spouse abuse to include violence perpetrated by women.

Identification of neural circuits by imaging coherent electrical activity with FRET-based dyes.

We show that neurons that underlie rhythmic patterns of electrical output may be identified by optical imaging and frequency-domain analysis. Our contrast agent is a two-component dye system in which changes in membrane potential modulate the relative emission between a pair of fluorophores. We demonstrate our methods with the circuit responsible for fictive swimming in the isolated leech nerve cord. The output of a motor neuron provides a reference signal for the phase-sensitive detection of changes in fluorescence from individual neurons in a ganglion. We identify known and possibly novel neurons that participate in the swim rhythm and determine their phases within a cycle. A variant of this approach is used to identify the postsynaptic followers of intracellularly stimulated neurons.

Photolysis of caged calcium in femtoliter volumes using two-photon excitation.

A new technique for the determination of the two-photon uncaging action cross section (deltau) of photolyzable calcium cages is described. This technique is potentially applicable to other caged species that can be chelated by a fluorescent indicator dye, as well as caged fluorescent compounds. The two-photon action cross sections of three calcium cages, DM-nitrophen, NP-EGTA, and azid-1, are studied in the range of excitation wavelengths between 700 and 800 nm. Azid-1 has a maximum deltau of approximately 1.4 GM at 700 nm, DM-nitrophen has a maximum deltau of approximately 0.013 GM at 730 nm, and NP-EGTA has no measurable uncaging yield. The equations necessary to predict the amount of cage photolyzed and the temporal behavior of the liberated calcium distribution under a variety of conditions are derived. These equations predict that by using 700-nm light from a Ti:sapphire laser focused with a 1.3-NA objective, essentially all of the azid-1 within the two-photon focal volume would be photolyzed with a 10-micros pulse train of approximately 7 mW average power. The initially localized distributions of free calcium will dissipate rapidly because of diffusion of free calcium and uptake by buffers. In buffer-free cytoplasm, the elevation of the calcium concentration at the center of the focal volume is expected to last for approximately 165 micros.

Laboratory-based measurements of swimming performance and related metabolic rates of field-sampled smallmouth buffalo (Ictiobus bubalus): a study of seasonal changes.

Numerous studies have demonstrated how the performance physiology of fish may change when they are acclimated to designated laboratory temperatures, but few researchers have examined naturally occurring seasonal effects on several physiological parameters associated with swimming performance. Using field-acclimatized smallmouth buffalo (Ictiobus bubalus) collected each season, we report significant seasonal effects in the following variables: critical swimming speed (modified), metabolic rate (standard, active, and scope for activity), and swimming efficiency (total and net cost of transport). Underlying seasonal changes in performance was the reproductive cycle of buffalo, particularly the period of fall gonadal recrudescence. Compared with spring, fall buffalo had a significantly lower mean critical swimming speed (72%) and lower active metabolic rate (53%), even when tested at similar temperatures. During spring, buffalo had a high mean critical swimming speed and low net cost of transport in comparison with other seasons. Buffalo are known to participate in a spring migration and spawning that may require the increased performance and efficiency observed during that season. In addition, significant sex effects were detected in winter measurements of standard metabolic rate and net cost of transport, with females the more efficient swimmers.

Specific covalent labeling of recombinant protein molecules inside live cells.

Recombinant proteins containing four cysteines at the i, i + 1, i + 4, and i + 5 positions of an alpha helix were fluorescently labeled in living cells by extracellular administration of 4',5'-bis(1,3, 2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.

A new caged Ca2+, azid-1, is far more photosensitive than nitrobenzyl-based chelators.

BACKGROUND: Photolabile chelators that release Ca2+ upon illumination have been used extensively to dissect the role of this important second messenger in cellular processes such as muscle contraction and synaptic transmission. The caged calcium chelators that are presently available are often limited by their inadequate changes in Ca2+ affinity, selectivity for Ca2+ over Mg2+ and sensitivity to light. As these chelators are all based on nitrobenzyl photochemistry, we explored the use of other photosensitive moieties to generate a new caged calcium with improved properties. RESULTS: Azid-1 is a novel caged calcium in which a fluorescent Ca2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca2+ with a dissociation constant (Kd) of approximately 230 nM, which changes to 120 microM after photolysis with ultraviolet light (330-380 nm). Mg2+ binding is weak (8-9 mM Kd) before or after photolysis. Azid-1 photolyzes with unit quantum efficiency, making it 40-170-fold more sensitive to light than caged calciums used previously. The photolysis of azid-1 probably releases N2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-withdrawing ability of the amidoxime cation reduces the chelator's Ca2+ affinity within at most 2 ms following a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in which Ca2+ photolytically released from azid-1 could replace the normal depolarization-induced Ca2+ transient in triggering synaptic plasticity. CONCLUSIONS: Azid-1 promises to be a useful tool for generating highly controlled spatial and temporal increases of Ca2+ in studies of the many Ca2+-dependent biological processes. Unlike other caged calciums, azid-1 has a substantial cross section or shows a high susceptibility for two-photon photolysis, the only technique that confines the photochemistry to a focal spot that is localized in three dimensions. Azide photolysis could be a useful and more photosensitive alternative to nitrobenzyl photochemistry.

Spatio-temporal dynamics of cyclic AMP signals in an intact neural circuitm.

The functional properties of neuronal networks can be reconfigured by a variety of modulatory neurotransmitters, which may alter the excitable properties of neurons or the strengths of synaptic connections. Many of these neuromodulators act via the intracellular second messenger cyclic AMP, but their effects on the spatial distribution of cAMP concentration have never been examined in an intact neural circuit. We therefore used the cAMP-indicator dye FICRhR (refs 1, 2) to investigate the effect of several neuromodulators (octopamine, dopamine, acetylcholine, serotonin and proctolin) on cAMP distribution in identified neurons of the lobster stomatogastric ganglion (STG). When added to the bath solution, each of these neuromodulators produced a unique pattern of cAMP transients among the different neurons of the STG. Electrical stimulation of neurons innervating the STG causes synaptic release of endogenous modulators, leading within a few seconds to local increases of cAMP in fine neurite branches, the site where many modulators are thought to act. After prolonged stimulation, cAMP diffuses from the site of production to throughout the neuritic tree and eventually to the cell body. Diffusion of cAMP may explain how transient localized inputs to a neuron can produce long-range effects such as long-term changes in gene expression.