Colocalization of fluorescent markers in confocal microscope images of plant cells

This protocol describes the steps needed to perform quantitative statistical colocalization on two-color confocal images, specifically of plant cells. The procedure includes a calibration test to check the chromatic alignment of the confocal microscope. A software tool is provided to calculate the Pearson and Spearman correlation coefficients ('Pearson-Spearman correlation colocalization' ImageJ plug-in) across regions of interest within the image. Steps are included to help the user practice using the software. The result is a quantitative estimate of the amount of colocalization in the images. Manual masking takes about 1-15 min per image, depending on the detail required, and calculating the correlation coefficients is almost instantaneous. Examples of suitable dyes for such two-color colocalization include Oregon Green or Alexa Fluor 488 dyes in the green range (excited with 488-nm laser line) and Alexa Fluor 555 dye in the red range (excited with 543-nm laser line).     

Characterization of G3BPs: tissue specific expression, chromosomal localisation and rasGAP(120) binding studies.

The G3BP (ras-GTPase-Activating Protein SH3-Domain-Binding Protein) family of proteins has been implicated in both signal transduction and RNA-metabolism. We have previously identified human G3BP-1, G3BP-2, and mouse G3BP-2. Here, we report the cloning of mouse G3BP-1, the discovery of two alternatively spliced isoforms of mouse, and human G3BP-2 (G3BP-2a and G3BP-2b), and the chromosomal localisation of human G3BP-1 and G3BP-2, which map to 5q14.2-5q33.3 and 4q12-4q24 respectively. We mapped the rasGAP(120) interactive region of the G3BP-2 isoforms and show that both G3BP-2a and G3BP-2b use an N-terminal NTF2-like domain for rasGAP(120) binding rather than several available proline-rich (PxxP) motifs found in members of the G3BPs. Furthermore, we have characterized the protein expression of both G3BP-1 and G3BP-2a/b in adult mouse tissues, and show them to be both tissue and isoform specific.     

Gene Transfer in Mycoplasma pulmonis

Experiments were undertaken to examine gene transfer in Mycoplasma pulmonis. Parent strains containing transposon-based tetracycline and chloramphenicol resistance markers were combined to allow transfer of markers. Two mating protocols were developed. The first consisted of coincubating the strains in broth culture for extended periods of time. The second protocol consisted of a brief incubation of the combined strains in a 50% solution of polyethylene glycol. Using either protocol, progeny that had acquired antibiotic resistance markers from both parents were obtained. Analysis of the progeny indicated that only the transposon and not flanking genomic DNA was transferred to the recipient cell. Gene transfer was DNase resistant and probably the result of conjugation or cell fusion.     

Endocrine Monitoring of Wild Dominant and Subordinate Female Leontopithecus rosalia

In captive callitrichid primates, female reproductive function tends to vary with social status. However, little is known about the interplay between these factors in wild groups. We report observations on normative ovarian function in dominant and subordinate female golden lion tamarins (Leontopithecus rosalia) living in wild groups. We monitored ovarian status by measuring, via enzyme immunoassay, concentrations of excreted pregnanediol glucuronide (PdG) and estrone conjugates (E₁C) in fecal samples collected noninvasively from individuals in social groups in the Poço das Antas Biological Reserve, Rio de Janeiro State, Brazil. Dominant breeding females demonstrated steroid levels similar to those previously reported for wild cotton-top tamarin females, with statistically significant rises during pregnancy. The duration of elevation of fecal steroids in breeding females was ca. 4 mo, which corresponds with estimates of gestation from captive studies. Low steroid concentrations from December to June suggest a seasonally-related period of infertility in female golden lion tamarins. Dominant and subordinate females demonstrated several differences in endocrine function. In general, younger females living in intact natal family groups showed no evidence of ovarian cyclicity. We noted endocrine profiles consistent with ovulation and subsequent pregnancy for behaviorally subordinate females living in groups with unrelated males or in which a reversal in female dominance status occurred. Results suggest that in addition to changes in female reproductive endocrinology associated with puberty, the regulation of reproduction in females in wild callitrichid groups can be sensitive to status and relatedness to breeding males.     

Slow adaptation in spider mechanoreceptor neurons

Slow adaptation of action potential firing is a common but poorly understood property of sensory neurons. We quantified slow adaptation in a cuticular mechanoreceptor organ of the spider, Cupiennius salei, by stimulating with continuous pseudorandom mechanical displacements while recording action potentials intracellularly from the cell bodies. Firing rate declined over a period of several minutes before reaching a steady level at about half the initial rate. This slow adaptation was fitted by an exponential decay with mean time constant of 18.5 s. Recovery from slow adaptation was also fitted by an exponential process, but with a longer time constant of 167 s. The receptor potential produced by the same stimulation protocol did not change its amplitude or dynamics, showing that slow adaptation occurs during action potential encoding from the receptor potential. Experiments with chemical blockers of calcium entry or the known potassium currents failed to reduce the slow adaptation. The Na⁺/K⁺ pump blocker Ouabain decreased the time constant of slow adaptation, suggesting that ion accumulation is involved. In some experiments, a second class of small action potentials were observed, which were tentatively attributed to failed conduction from the sensory dendrite through the soma to the axon.     

Trans-channel interactions in batrachotoxin-modified skeletal muscle sodium channels: voltage-dependent block by cytoplasmic amines, and the influence of mu-conotoxin GIIIA derivatives and permeant ions

External μ-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two μ-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the μ-conotoxin and the DEA-binding site of ∼15 Å. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by ∼4-fold; and 2), increasing external [Na⁺] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions.     

Weight‐Bearing Physical Activity among Girls and Mothers: Relationships to Girls’ Weight Status

Objective: To assess weight‐bearing physical activity (WBPA) barriers, benefits, self‐efficacy, social influence, and behaviors [WBPA and physical activity (PA)] among girls and their mothers according to girls’ weight status (nonoverweight vs. overweight). Research Methods and Procedures: Participants were 9‐ to 11‐year‐old girls (n = 295) and their mothers who participated in the baseline assessment of a nutrition and PA intervention trial. Girls’ and mothers’ WBPA attitudes and mothers’ WBPA behaviors were self‐reported on questionnaires. Girls’ WBPA and total PA behaviors were self‐reported using a structured interview (Physical Activity Checklist Interview). Stature and weight were measured by standardized anthropometrics. Overweight status was based on BMI. Results: Compared with nonoverweight girls, overweight girls were significantly more likely to report barriers to WBPA participation and perceive social influence from family and friends to do more WBPA. They were also significantly less likely to report self‐efficacy regarding WBPA and to believe that they did enough WBPA. Compared with mothers of nonoverweight girls, mothers of overweight girls were significantly more likely to report that it is difficult to persuade their daughters to do more WBPA and significantly less likely to report that WBPA was fun for their daughters. Girls’ overweight status was not associated with girls’ reports of minutes spent per week in PA or WBPA. Discussion: The present study's findings of lower WBPA self‐efficacy, lack of enjoyment of WBPA, and higher perceived social influence to do WBPA among overweight girls suggest that efforts are needed to promote physical competencies and positive perceptions of PA among overweight girls.     

Large-scale transposon mutagenesis of Mycoplasma pulmonis

To obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon Tn 4001T was determined for 1700 members of a library of Mycoplasma pulmonis mutants. After evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. The dispensable and essential genes of M. pulmonis were compared with those reported for Mycoplasma genitalium and Bacillus subtilis . Perhaps the most surprising result of the current study was that unlike other bacteria, ribosomal proteins S18 and L28 were dispensable. Carbohydrate transport and the susceptibility of selected mutants to UV irradiation were examined to assess whether active transposition of Tn 4001T within the genome would confound phenotypic analysis. In contrast to earlier reports suggesting that mycoplasmas were limited in their DNA repair machinery, mutations in recA , uvrA , uvrB and uvrC resulted in a DNA-repair deficient phenotype. A mutant with a defect in transport of N -acetylglucosamine was identified.