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Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers.

Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei.

Cell Line↗

A practical approach in the treatment of self-inflicted violence.

After a brief review of the literature on self-inflicted violence, a practical approach to the treatment of patients who show self-injurious behavior and self-mutilation is developed. The treatment model uses therapeutic interventions based on a biopsychosocial understanding of the person and applies knowledge from the biological, psychological-behavioral, and social fields. Interventions discussed include ego strengthening, planning for substitute behaviors, therapeutic imagery, ego-state therapy, reframing, cognitive syllogism, behavioral reinforcement and extinction, and use of behavioral contracts. The case examples presented illustrate the importance of the therapist/patient relationship, which is based on trust, respect, and caring but also on patient responsibility for change.

Adult↗

Cystine/glutamate exchange regulates metabotropic glutamate receptor presynaptic inhibition of excitatory transmission and vulnerability to cocaine seeking.

Withdrawal from chronic cocaine reduces extracellular glutamate levels in the nucleus accumbens by decreasing cystine/glutamate exchange (xc-). Activating xc- with N-acetylcysteine restores extracellular glutamate and prevents cocaine-induced drug seeking. It was hypothesized that the activation of xc- prevents drug seeking by increasing glutamatergic tone on presynaptic group II metabotropic glutamate receptors (mGluR2/3) and thereby inhibiting excitatory transmission. In the first experiment, the capacity of glutamate derived from xc- to regulate excitatory transmission via mGluR2/3 was determined. Physiological levels of cystine (100-300 nm) were restored to acute tissue slices from the nucleus accumbens or prefrontal cortex. Cystine increased glutamate efflux and decreased miniature EPSC (mEPSC) and spontaneous EPSC (sEPSC) frequency as well as evoked EPSC amplitude. These effects of cystine were presynaptic, because there was no change in mEPSC or sEPSC amplitude, and an increase in the evoked EPSC paired-pulse facilitation ratio. The cystine-induced reduction in EPSCs was reversed by blocking either xc- or mGluR2/3. In the second experiment, blocking mGluR2/3 prevented the ability of N-acetylcystine to inhibit the reinstatement of drug seeking in rats trained to self-administer cocaine. These data demonstrate that nonsynaptic glutamate derived from xc- modulates synaptic glutamate release and thereby regulates cocaine-induced drug seeking.

Amino Acids↗

Interaction of human cathepsin D with the inhibitor pepstatin.

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.

Binding Sites↗

A condition for the extinction of a branching process with an absorbing lower barrier.

A branching process with an absorbing lower barrier is considered. This is a Galton-Watson process with the condition that at any generation the number of individuals is greater than a lower barrier or it is equal to zero (i.e. all individuals in populations which are too small die and have no offspring). A necessary and sufficient condition is given for the process to become extinct with probability one. At the end of the paper there are three illustrating examples.

Models, Biological↗

A parity-dependent immigration-birth-death-emigration process.

An immigration-birth-death-emigration process is studied when the immigration, birth, death, and emigration parameters depend on two parity classes of individuals, namely, zero and nonzero parity. The expected number of individuals in the population at time t and that of emigrants in (0,t) of both parity classes are obtained using the probability generating function (pgf) technique. The explicit expression for the pgf of the process is derived when the parameters are parity-independent. Population extinction and some limiting results are also discussed.

Candida albicans↗

The attractiveness of the Droop equations.

The Droop equations are a system of three coupled, nonlinear ordinary differential equations describing the growth of a microorganism in a chemostat. The growth rate of the organism is limited by the availability of a single nutrient. In contrast to the better known Monod equations, the nutrient is divided into external and internal cellular pools. Only the internal pool can catalyze growth. This paper proves that the Droop equations are globally stable. Based on a single combination of parameters, either the chemostat organism goes extinct or it tends to a fixed, positive concentration.

Bacteria↗

Extinction times for a birth-death process with two phases.

Many populations have a negative impact on their habitat or upon other species in the environment if their numbers become too large. For this reason they are often subjected to some form of control. One common control regime is the reduction regime: when the population reaches a certain threshold it is controlled (for example culled) until it falls below a lower predefined level. The natural model for such a controlled population is a birth-death process with two phases, the phase determining which of two distinct sets of birth and death rates governs the process. We present formulae for the probability of extinction and the expected time to extinction, and discuss several applications.

Animals↗

Analysis of a delayed epidemic model with pulse vaccination and saturation incidence.

Pulse vaccination is an important strategy for the elimination of infectious diseases. An SEIRS epidemic model with time delays and pulse vaccination is formulated in this paper. Using the discrete dynamical system determined by the stroboscopic map, we obtain the exact infection-free periodic solution of the impulsive epidemic system and prove that the infection-free periodic solution is globally attractive if the vaccination rate is larger than theta*. Moreover, we show that the disease is uniformly persistent if the vaccination rate is less than theta*. The permanence of the model is investigated analytically. Our results indicate that a long latent period of the disease is sufficient condition for the extinction of the disease.

Disease Outbreaks↗

A computer exploration of some properties of non-linear stochastic partnership models for sexually transmitted diseases with stages.

In this paper, branching process approximations to non-linear stochastic partnership models for sexually transmitted diseases in heterosexual populations were used to find points in the parameter space such that an epidemic would occur. At selected points in the parameter space, samples of Monte Carlo realizations of the process were computed and analyzed statistically to gain insights into the stochastic evolution of epidemics seeded by one infective single female and male. Non-linear difference equations were embedded in the stochastic processes, making it possible to compare trajectories computed according to the deterministic model with those computed from samples of Monte Carlo realizations. From these trajectories it was shown that stochastic fluctuations may have a profound effect on the long-term evolution of an epidemic, and examples demonstrate that an investigator may be misled if a deterministic model alone were used to project an epidemic, particularly when there is a significant probability of extinction.

Computer Simulation↗

Life-history models of extinction: a test with island spiders.

This study analyzes extinction patterns for two species of orb spiders monitored annually on 77 islands over a continuous 20-yr period. One species, Argiope argentata, has large populations sometimes crashing quickly to extinction and a much weaker relation of extinction likelihood to population size than does the other species, Metepeira datona. Demographic models were built for both species and matched against observations. Differences between the species in life-history traits-estimated with measurements from the field-together with incorporation of demographic stochasticity, a population ceiling, and environmental stochasticity, were necessary to fit the observed extinction curves. As predicted from life-history patterns, long-term population growth rates (and hence predicted extinction probabilities) are relatively very sensitive to values of juvenile survivorship. Models are also sensitive to variation in the population ceiling and environmental noise, which tend to act in a complementary manner. A simple model with no age structure was able to fit the data on large initial population sizes but not on small initial population sizes, showing that life cycle characteristics interact with the various sources of stochasticity and hence have to be taken into account to produce a precise model of the extinction process.

Age Factors↗

Landscape history, fragmentation, and patch occupancy: models for a forest bird with limited dispersal.

We developed and tested patch occupancy models for an endemic understory bird with limited dispersal ability, the Chucao Tapaculo (Scelorchilus rubecula), in two South American temperate rain forest landscapes that differed in levels and duration of forest loss. We assessed cover changes since 1961 in each landscape and surveyed patches for Chucao Tapaculo occupancy. We then developed incidence-based predictive models independently for each landscape and tested each model reciprocally in the alternative study area. We thereby assessed the domain of model applicability and identified those predictor variables with general effects and those that varied between the two landscapes. The two models were consistent regarding variable selection, and predictive accuracy of each model was high in the landscape where training data were collected. However, the models differed substantially in the magnitudes of effects related to patch size, with larger unoccupied patches observed in the landscape with the more advanced stage of fragmentation. Due to this discrepancy, each model performed poorly when applied to the alternative landscape, potentially reflecting the contrasting stages of habitat loss. Although it was impossible to dissociate effects of level and duration of forest loss, we viewed the landscapes as representing two extremes along a continuum of fragmentation, providing insights into potential trajectories for portions of the biome where deforestation is occurring. Further, our data suggest that static equilibrium models developed from distribution patterns in recently fragmented landscapes may overestimate persistence when used as a forecasting tool, or when extrapolated to alternative landscapes where fragmentation is more advanced. Thus, we suggest that landscapes used as standards for model building should be selected with caution. We recommend that distribution patterns be obtained from landscapes where fragmentation is advanced, preferably with histories of fragmentation long enough that time-delayed extinctions already would have occurred.

Animals↗

An intraspecific comparative analysis of character divergence between sympatric species.

Although sympatric character divergence between closely related species has been described in a wide variety of taxa, the evolutionary processes responsible for generating these patterns are difficult to identify. One hypothesis that can explain sympatric differences is ecological character displacement: the sympatric origin of morphologically divergent phenotypes in response to selection caused by interspecific competition. Alternatively, populations may adapt to different conditions in allopatry, with sympatric distributions evolving through selective colonization and proliferation of ecologically compatible phenotypes. In this study, I characterize geographic variation within two sibling species of rocky-shore gastropods that have partially overlapping distributions in central California. In sympatry, both Nucella emarginata and N. ostrina show significant differences in shell shape and shell ornamentation that together suggest that where the two species co-exist, divergent phenotypes arose as an evolutionary consequence of competition. To examine the evolutionary origins of divergent characters in sympatry, I used a comparative method based on spatial autocorrelation to remove the portion of the phenotypic variance among populations that is explained by genetic distance (using mitochondrial DNA sequences and allozyme frequency data). Because the remaining portion of the phenotypic variance represents the independent divergence of individual populations, a significant sympatric difference in the corrected dataset provides evidence of true character displacement: significant sympatric character evolution that is independent of population history. After removal of genetic distance effects in Nucella, shell shape differences remain statistically significant in N. emarginata, providing evidence of significant sympatric character divergence. However, for external shell ornamentation in both species and shell shape in N. ostrina, the significance of sympatric differences is lost in the corrected dataset, indicating that colonization events and gene flow have played important roles in the evolutionary history of character divergence in sympatry. Although the absence of a widely dispersing planktonic larva in the life cycle of Nucella will promote local adaptation, the results here indicate that once advantageous traits arise, demographic processes, such as recurrent gene flow between established populations and extinction and recolonization, are important factors contributing to the geographic pattern of sympatric character divergence.

Animals↗

Quantitative estimation of the degree of cell spreading on different surfaces and cell monolayers using a fluorescent plate scanner.

A simple, rapid, and quantitative method for estimation of the degree of spreading of fluorescently labeled cells adherent to an artificial or biological surface has been designed, based on the principle that the medium between adherent cells and the surface of adhesion absorbs fluorescent light emitted by the adherent cells. Using the Beer-Lambert equation for extinction coefficient of monochromatic light, we have calculated the factor by which fluorescence signal actually emitted from cells adherent to the bottom of the microtiter well would differ from the fluorescence of these cells detected by a bottom-scanning plate reader. The physical nature of cell-cell ligand-receptor bridges depends on many parameters, such as the type of the receptor(s), the intensity and direction of a stress force, blockade of the receptors with monoclonal antibodies (MAb), and transfer of adhesion mediation to different ligand-receptor pairs. When employed in various adhesion models, this new technique has demonstrated quantitative changes of cellular spreading of human neutrophils adherent to different extracellular matrix protein-coated surfaces and to endothelial cells after neutrophil/endothelial cell activation or in the presence of MAb directed against neutrophil integrins. These measurements of spreading of adherent cells appear more sensitive than visual observation of cellular morphology on biological surfaces or cells.

Cell Adhesion↗

Problems in Extinction Model Selection and Parameter Estimation.

It is a vexing problem to achieve a consensus about the proper scientific way to assess population viability for habitat conservation plans. Rather than a hypothesis-testing approach, here it is proposed to select population models, estimate extinction parameters, and assess prediction uncertainty using a pragmatic, empirical Bayesian approach. The simplest usable models include the effects of population growth, r; carrying capacity, K; Allee threshold, N(A); and environmental stochasticity, v(r). Analytic predictions of expected extinction times are available for such models. Models that are more complex can be elaborated from this basis. Selection from a hierarchy of nesting population models can often be done through the evaluation of parameters. The estimation of the most important extinction parameters can be undertaken in a variety of ways. Time series can be analyzed to estimate r(d), v(r), rho, and K. Habitat models and individualistic population models may help estimate N(A) and K and demographic stochasticity. Fine-scale biogeography and climatological data may be useful in the estimation of a variety of parameters. Because it takes many years to estimate extinction parameters accurately for a given population of interest, the most efficient estimation procedures are desirable. I propose the use of prior information from an (as yet nonexistent) population biology database. The accumulation of local information through monitoring will improve our estimates allowing adaptive management. Uncertainty in the estimates will always remain, but it may be quantified by the posterior distributions. A crude example is discussed using treefrog population data. Although the motivations, beliefs, and biases of competing stakeholders will differ, a habitat conservation plan could accommodate this variation in the prior distributions. Field experience from monitoring will increasingly clear up any discrepancies between the opposing beliefs and the real ecosystem. As the world is an uncertain place and because there is no universal scientific method, there will always be controversy and surprises. The best we can do is (1) agree about our prior information, (2) agree about the strategy of model selection and parameter estimation, and (3) agree about our strategy for adaptive management. Perhaps the greatest impediment to such prior agreements for HCPs is the likely paranoia inspired by the use of unfamiliar statistical methodology. We need to train students of ecology in a more flexible and deeper understanding of statistics and philosophy of science.

Journal Article↗