Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed.

Prospecting and dispersal: their eco-evolutionary dynamics and implications for population patterns.

Dispersal is not a blind process, and evidence is accumulating that individual dispersal strategies are informed in most, if not all, organisms. The acquisition and use of information are traits that may evolve across space and time as a function of the balance between costs and benefits of informed dispersal. If information is available, individuals can potentially use it in making better decisions, thereby increasing their fitness. However, prospecting for and using information probably entail costs that may constrain the evolution of informed dispersal, potentially with population-level consequences. By using individual-based, spatially explicit simulations, we detected clear coevolutionary dynamics between prospecting and dispersal movement strategies that differed in sign and magnitude depending on their respective costs. More specifically, we found that informed dispersal strategies evolve when the costs of information acquisition during prospecting are low but only if there are mortality costs associated with dispersal movements. That is, selection favours informed dispersal strategies when the acquisition and use processes themselves were not too expensive. When non-informed dispersal strategies evolve, they do so jointly with the evolution of long dispersal distance because this maximizes the sampling area. In some cases, selection produces dispersal rules different from those that would be 'optimal' (i.e. the best possible population performance--in our context quantitatively measured as population density and patch occupancy--among all possible individual movement rules) for the population. That is, on the one hand, informed dispersal strategies led to population performance below its highest possible level. On the other hand, un- and poorly informed individuals nearly optimized population performance, both in terms of density and patch occupancy.

Risky movement increases the rate of range expansion.

The movement rules used by an individual determine both its survival and dispersal success. Here, we develop a simple model that links inter-patch movement behaviour with population dynamics in order to explore how individual dispersal behaviour influences not only its dispersal and survival, but also the population's rate of range expansion. Whereas dispersers are most likely to survive when they follow nearly straight lines and rapidly orient movement towards a non-natal patch, the most rapid rates of range expansion are obtained for trajectories in which individuals delay biasing their movement towards a non-natal patch. This result is robust to the spatial structure of the landscape. Importantly, in a set of evolutionary simulations, we also demonstrate that the movement strategy that evolves at an expanding front is much closer to that maximizing the rate of range expansion than that which maximizes the survival of dispersers. Our results suggest that if one of our conservation goals is the facilitation of range-shifting, then current indices of connectivity need to be complemented by the development and utilization of new indices providing a measure of the ease with which a species spreads across a landscape.

Cyclic voles and shrews and non-cyclic mice in a marginal grassland within European temperate forest.

Cyclic population dynamics of small mammals are not restricted to the boreal and arctic zones of Eurasia and North America, but long-term data series from lower latitudes are still less common. We demonstrated here the presence of periodic oscillations in small mammal populations in eastern Poland using 22-year (1986-2007) trapping data from marginal meadow and river valley grasslands located in the extensive temperate woodland of Bialowieza Primeval Forest. The two most common species inhabiting meadows and river valleys, root vole Microtus oeconomus and common shrew Sorex araneus, exhibited synchronous periodic changes, characterised by a 3-year time lag as indicated by an autocorrelation function. Moreover, the cycles of these two species were synchronous within both habitats. Population dynamics of the striped field mouse Apodemus agrarius was not cyclic. However, this species regularly reached maximum density 1 year before the synchronized peak of root voles and common shrews, which may suggest the existence of interspecific competition. Dynamics of all three species was dominated by direct density-dependent process, whereas delayed density dependent feedback was significant only in the root vole and common shrew. Climatic factors acting in winter and spring (affecting mainly survival and initial reproduction rates) were more important than those acting in summer and autumn and affected significantly only the common shrew. High temperatures in winter and spring had positive effects on autumn-to-autumn changes in abundance of this species, whereas deep snow in combination with high rainfall in spring negatively affected population increase rates in common shrew.

Prospects in plant genetic engineering.

The functional expression of a novel gene in a genetically engineered plant has not yet been reported. One major barrier in movement toward this goal is our limited understanding of the molecular bases of gene expression. Attempts to establish genetic engineering as a practical facet of plant breeding are also complicated by the fact that genes for most important plant characteristics have not yet been identified. However, the benefits to be gained from all aspects of plant improvement are stimulating research into both the development of plant transformation technology and the isolation and characterization of genes responsible for valuable traits. As scientists develop greater knowledge of plant molecular genetics, we can expect to see practical applications in such diverse areas as improvement of plant nutritional quality, decreases in fertilization requirements, and increases in resistance to environmental stresses and pathogens.

Antecedents and consequences of acute stress disorder among motor vehicle accident victims.

The aim of this study was to examine differences between motor vehicle accident (MVA) victims diagnosed retrospectively with Acute Stress Disorder (ASD) and subsequent Post-Traumatic Stress Disorder (PTSD), and MVA victims with only PTSD. A total of 14 ASD subjects and 28 PTSD subjects (matched on age and on sex) were compared on subjects' levels of functioning and symptom severity at three time-points: pre-MVA, 1-4 months post-MVA (initial assessment), and 6 months post initial assessment. Fourteen non-MVA subjects were included as controls in the analyses for pre-MVA differences and presence of psychopathology at time of initial assessment. ASDs had higher rates of previous mood disorders (other than major depression), previous AXIS-I disorders, and previous AXIS-II disorders than did PTSDs. No differences existed between the groups at 6-month follow-up. These results provide clinically useful information regarding characteristics that predispose people to develop ASD in the aftermath of a trauma. Previous research with trauma victims has demonstrated dissociative symptoms to be associated with higher PTSD symptom severity and longer symptom duration, but our follow-up results suggest that ASD, which is characterized by dissociative symptomatology, is not indicative of poorer outcome in MVA victims.

One-year prospective follow-up of motor vehicle accident victims.

One-hundred and thirty-two victims of motor vehicle accidents (MVAs), who sought medical attention as a result of the MVA, were assessed at three points in time: 1-4 months post-MVA, 6 months later, and 12 months later. Of the 48 who met the full criteria for Post-Traumatic Stress Disorder (PTSD) initially, half had remitted at least in part by the 6-month follow-up point and two-thirds had remitted by the 1-yr follow-up. Using logistic regression, 3 variables combined to correctly identify 79% of remitters and non-remitters at the 12-month follow-up point: initial scores on the irritability and foreshortened future symptoms of PTSD and the initial degree of vulnerability the subject felt in a motor vehicle after the MVA. Four variables combined to predict 64% of the variance in the degree of post-traumatic stress symptoms at 12 months: presence of alcohol abuse and/or an Axis-II disorder at the time of the initial assessment as well as the total scores on the hyperarousal and on avoidance symptoms of PTSD present at the initial post-MVA assessment.

Psychometric properties of a measure of aggressive driving: the Larson Driver's Stress Profile.

This paper presents three studies on Larson's 1996 Driver's Stress Profile, a measure of aggressive driving tendencies. In Study 1, utilizing 33 individuals (15 men, 18 women) who took the test twice with one week between tests, we found the test to have good test-retest reliability (r = .93). In Study 2, utilizing 176 individuals (77 men, 99 women), we found different preliminary norms for men and women and good internal consistency (.93). We also found significant correlation between the total test scores and age (r = -.27) and a significant correlation (age corrected) between total test scores and number of self-reported MVAs (r = .28). An exploratory factor analysis indicated that three factors, including 28 of the 40 items, accounted for 43.4% of the variance. In study 3, utilizing the same subjects as in Study 2 (84 individuals from the community of average age 35.3 yr.; 92 college students of average age 18.9 yr.), we found the full scale scores correlated significantly with scores on Trait Anger and Anger Out and Type A Behavior, especially speed and impatience. Finally, scores correlated significantly with the Deffenbacher Driving Anger Scale (r = .57) but clearly tapped processes different, in part, from those measured by the Deffenbacher, et al. scale (1994). Overall, the Driver's Stress Profile appears to be a sound, reliable, and valid scale for use with aggressive driving.

The failure of intensive self-regulatory treatment with chronic daily headache: a prospective study.

Sixteen patients with chronic daily headache (HA) of moderate to severe intensity received 20 sessions of self-regulatory treatment including progressive muscle relaxation, thermal biofeedback, and cognitive stress coping therapy. Four stopped treatment after 12 visits (but had received all 3 treatment components). Only 2 of 12 completers (17%) showed more than 50% reduction in HA Index based on daily diaries. Chronic daily HA continues to be relatively refractory to self-regulatory treatment.

Cytokinin antagonist activity of substituted phenethylamines in plant cell culture.

A series of structurally related substituted phenethylamines shows extreme toxicity toward wild-type callus tissue cultures of tobacco (Nicotiana tabacum), soybean (Glycine max), corn (Zea mays), and sunflower (Helianthus annuus L.), but tobacco crown gall cultures are resistant to the compounds. The essential components that result in toxicity of the phenethylamines include one aromatic hydroxyl and one primary aliphatic amino group. A series of attenuated crown gall cultures, generated by transformation of tobacco with various modified Agrobacterium strains, has been used to demonstrate that the resistance of crown galls to the phenethylamines is due to the expression in these tissues of isopentenyl transferase, a first step in cytokinin biosynthesis. The toxicity of the compounds to tissue cultures is very rapid, but can be overcome by prior exposure of the calli to exogenous cytokinin. Because of the relationships we have observed between cytokinins and these compounds, we propose that the substituted phenethylamines may represent a class of chemicals that can be used as specific probes to further an understanding of cytokinin metabolism in plant tissues.

Biosynthesis of subunits of the soybean 7S storage protein.

Biosynthesis of the alpha', alpha, and beta subunits of the soybean 7S storage protein (conglycinin) was studied by in vivo and in vitro experiments. One hour in vivo labeling produced polypeptides of 72 kilodaltons (kD) and 78 kD which, in a subsequent 1-h chase period, gave rise to a heterodisperse band of polypeptides of 76-83 kD, the apparent molecular weights of mature alpha and alpha', respectively. After a 3-h chase period only mature alpha and alpha' were labeled. The pre-alpha' (80 kD) and pre-alpha (78 kD) polypeptides produced by in vitro translation of total seed poly(A)+ RNA did not coelectrophorese with polypeptides labeled in vivo. The mature beta-subunit (53 kD), produced in vivo during the 1-h chase period, apparently was translated as a 50 kD polypeptide in vitro. The data suggest that, in vivo, primary translation products undergo both cotranslational and posttranslational modifications during the formation of mature subunits. cDNA clones complementary to soybean seed poly(A)+ RNA were prepared and subsequently identified by hybrid-release and immunoprecipitation experiments. Clone pGmc 236 (550 base pairs) (bp) was shown to contain sequences complementary to mRNAs for the alpha', alpha, and beta subunits on the basis of hybrid-release experiments, corroborating tryptic fingerprints that demonstrate that these subunits are closely related to each other. Although selected by a single cDNA clone, the mRNAs are not identical because the alpha' mRNA was eluted from the filter-bound cDNAs at a lower temperature than were the alpha beta mRNAs, pGmc 236 hybridized on Northern blots to mRNAs of approximately 2500 nucleotides, sufficient size to code for the alpha' or alpha subunit of the 7S storage protein.

Bacillus thuringiensis section sign-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects.

The crystal proteins, or section sign-endotoxins, of Bacillus thuringiensis are specifically lethal to Lepidopteran insects. We utilized a truncated and modified portion of a cloned crystal protein gene to construct a chimeric gene capable of expression in plant cells. Using an Agrobacterium tumefaciens binary vector system, we then transferred the chimeric toxin gene into tobacco (Nicotiana tabacum cv Havana 425) cells and regenerated recombinant plants. One to several copies per cell of the toxin gene are routinely present in the recombinant plants. Hybridization experiments demonstrated that these plants had a new RNA species of the size expected for the truncated toxin mRNA, and a polypeptide having the mobility expected for the truncated toxin was detected by immunoblotting. Significant variation was found in the levels of toxin-specific RNA expression between different recombinants, but the levels of hybridizing RNA in transformants correlated with the level of toxicity demonstrated against Manduca sexta (tobacco hornworm), and other Lepidopteran insects. The recombinant genes were transmitted to progeny and resistance to insects was maintained, thus demonstrating that the introduction of toxin genes into plants may be a practical method of providing protection against certain insect pests.

Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny.

Cloned DNA sequences encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase have been inserted into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the "rooty" locus. Transformation of tobacco stem segments with the engineered bacterial strains produced attenuated crown gall tumors that were capable of regeneration into intact, normal tobacco plants. The yeast gene and entire transferred DNA (T-DNA) were present in the regenerated plants in multiple copies, and nopaline was found in all tissues. The plants were fertile, and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA. Expression of nopaline in the germinated seedlings derived from one regenerated plant was variable and did not correlate with the levels of T-DNA present in the seedlings. Preliminary evidence indicates that nopaline in progeny of other similarly engineered plants is more uniform. The disarming of pTiT37 by insertions at the "rooty" locus thus appears to produce a useful gene vector for higher plants.

Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration.

Three different target tissues (protoplast-derived cells, nodules, and stems) and two unrelated hybrid genotypes of Populus (P. alba x P. grandidentata 'Crandon' and P. nigra 'Betulifolia' x P. trichocarpa) have been stably transformed by electric discharge particle acceleration using a 18.7 kb plasmid containing NOS-NPT, CaMV 35S-GUS, and CaMV 35S-BT. Four transformed plants of one hybrid genotype, NC5339, containing all 3 genes were recovered and analyzed. Two expressed GUS and one was highly resistant to feeding by 2 lepidopteran pests (the forest tent caterpillar, Malacosoma disstria, and the gypsy moth, Lymantria dispar.) Pretreatment of the target tissues, fine-tuning of the bombardment parameters, and the use of a selection technique employing flooding of the target tissues were important for reliable recovery of transformed plants.

Surgical results in malignant glaucoma refractory to medical or laser therapy.

We investigated the surgical results in 25 patients identified with malignant (ciliary block) glaucoma. Four of these patients (16%) had successful outcomes following laser treatment alone. Nineteen patients (76%) underwent microsurgery for glaucoma refractory to conventional medical and/or laser therapy and are the subjects of this study. Core vitrectomy was successful in 4 of 6 pseudophakic patients (67%) and in 1 of 4 phakic patients (25%). Combined cataract extraction and vitrectomy had a higher success rate in patients undergoing posterior capsulectomy at the time of surgery (5 of 6 patients; 83%) compared with those patients left with an intact posterior capsular bag (1 of 4 patients; 25%). Intracapsular cataract extraction was successful in 1 of 2 patients (50%). We conclude that surgical vitrectomy in the presence of an intact posterior capsule may preclude the surgical resolution of aqueous misdirection. In eyes with pre-existing cataract, we recommend combined lens extraction, primary posterior capsulectomy and surgical vitrectomy for the surgical management of phakic malignant glaucoma.

The biosynthesis and processing of high molecular weight precursors of soybean glycinin subunits.

The predominant storage protein of soybean seed, glycinin, is composed of two heterogeneous classes of related subunits, the acidics (Mr approximately 38,000) and the basics (Mr approximately 22,000). Immunoreaction of polypeptides translated in vitro from isolated seed mRNA using antibodies prepared against either purified acidic or basic subunit groups precipitated precursor polypeptides of Mr = 60,000 to Mr = 63,000. High pressure liquid chromatography fingerprinting of trypsin-generated fragments from in vitro synthesized precursors showed fragments specific to both acidic and basic subunits. No mature acidic or basic subunits were detected in vitro translation reactions by either immunoprecipitation or high pressure liquid chromatography fingerprinting. Pulse-labeling of cotyledons growing in culture with [3H]glycine showed rapid accumulation of label in glycinin precursors of Mr = 59,000 to Mr = 62,000. Although in vivo synthesized precursors had slightly greater electrophoretic mobility than in vitro synthesized precursors, little label initially appeared in mature glycinin subunits. After several hours of continued cotyledon growth in absence of label, precursors were processed and label accumulated in both acidic and basic subunit groups. Recombinant plasmids were prepared by reverse transcription of soybean seed mRNA, and clones which encode glycinin precursors were identified by heteroduplex-hybridization of translatable messages. Northern blot analysis of seed mRNA shows the mRNA-encoding glycinin precursors to migrate at Mr = 0.71 X 10(6) on agarose gels, corresponding to approximately 2050 nucleotides. This is sufficiently large to encode a polypeptide consisting of both a glycinin acidic and basic subunit.