Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.     

Mitochondrial DNA analysis of Rhipicephalus sanguineus s.l. from the western Iberian peninsula

Rhipicephalus sanguineus Latreille (1806) (Ixodida: Ixodidae) is considered to be the most widely distributed tick and to have a vast range of habitats and hosts, including livestock, pets and wildlife. In addition to morphological differences, recent investigations using approaches based on molecular genetic markers have revealed the existence of different R. sanguineus lineages in different geographic regions. In this study, 475 ticks collected from dogs in the western Iberian peninsula were studied both morphologically and genetically, using 12S and 16S rDNA and COI gene markers in order to clarify the controversy over the systematic status of R. sanguineus sensu lato in Western Europe, and to compare the present data with those sourced from studies conducted in other regions of the world. Despite the high morphometric variability, particularly on spiracles in both genders and in female genitalia, data obtained with different genetic molecular markers show very low variability, suggesting the existence of a unique species. In addition, the phylogenetic analysis showed genetic uniformity, supporting the existence of a well‐defined clade consisting of R. sanguineus s.l. specimens from Western Europe that are distinct from R. sanguineus s.l. from Africa. Furthermore, these data corroborate the existence of a polymorphic species in Western Europe, which requires to be consensually redescribed in view of its medical and veterinary importance in pathogen transmission.     

Isolation of the fourth component (C4) of rat complement.

The fourth component of rat complement was purified to homogeneity by sequential chromatography of rat plasma in benzamidine on QAE-A50, SP-C50, hydroxyapatite, and gel filtration on Bio-Gel A 1.5. The final material was homogeneous on SDS-PAGE analysis and had a calculated m.w. of 198,000. A monospecific antibody against rat C4 was obtained from immunized rabbits. The concentration of rat C4 in the plasma of normal 4-month-old Wistar rats was 190 +/- 34 microgram/ml (mean +/- 1 S.D.).