Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus

Sulfated polysaccharides (SPs) extracted from five seaweed samples collected or cultivated in Mexico (Macrocystis pyrifera,Eisenia arborea,Pelvetia compressa,Ulva intestinalis, andSolieria filiformis) were tested in this study in order to evaluate their effect on measles virusin vitro. All polysaccharides showed antiviral activity (as measured by the reduction of syncytia formation) and low cytotoxicity (MTT assay) at inhibitory concentrations. SPs fromEisenia arboreaandSolieria filiformisshowed the highest antiviral activities (confirmed by qPCR) and were selected to determine their combined effect. Their synergistic effect was observed at low concentrations (0.0274 μg/mL and 0.011 μg/mL ofE. arboreaandS. filiformisSPs, resp.), which exhibited by far a higher inhibitory effect (96% syncytia reduction) in comparison to the individual SP effects (50% inhibition with 0.275 μg/mL and 0.985 μg/mL ofE. arboreaandS. filiformis, resp.). Time of addition experiments and viral penetration assays suggest that best activities of these SPs occur at different stages of infection. The synergistic effect would allow reducing the treatment dose and toxicity and minimizing or delaying the induction of antiviral resistance; sulfated polysaccharides of the tested seaweed species thus appear as promising candidates for the development of natural antiviral agents.

Asymmetric cell division in fucoid algae: a role for cortical adhesions in alignment of the mitotic apparatus

The first cell division in zygotes of the fucoid brown alga Pelvetia compressa is asymmetric and we are interested in the mechanism controlling the alignment of this division. Since the division plane bisects the mitotic apparatus, we investigated the timing and mechanism of spindle alignments. Centrosomes, which give rise to spindle poles, aligned with the growth axis in two phases – a premetaphase rotation of the nucleus and centrosomes followed by a postmetaphase alignment that coincided with the separation of the mitotic spindle poles during anaphase and telophase. The roles of the cytoskeleton and cell cortex in the two phases of alignment were analyzed by treatment with pharmacological agents. Treatments that disrupted cytoskeleton or perturbed cortical adhesions inhibited pre-metaphase alignment and we propose that this rotational alignment is effected by microtubules anchored at cortical adhesion sites. Postmetaphase alignment was not affected by any of the treatments tested, and may be dependent on asymmetric cell morphology.

F-actin marks the rhizoid pole in living Pelvetia compressa zygotes

Spatial and temporal changes in F-actin during polarity establishment in Pelvetia compressa zygotes were investigated using vital staining with rhodamine phalloidin (RP). F-actin was localized to a patch in the cortex of young zygotes. When unilateral light was applied to induce a growth axis (photopolarization) in a population of zygotes, the cortical F-actin patches localized at the shaded pole (rhizoid pole of growth axis). Treatments that prevented photopolarization prevented localization of F-actin patches to the shaded pole. When the direction of the light treatment was reversed, the previous growth axis was abandoned and a new axis was established in the opposite direction. The F-actin patch repositioned to the new rhizoid pole within minutes of light reversal, indicating that F-actin was an immediate marker of the nascent growth axis. Repositioning probably occurred by disassembly of the initial patch and reassembly of a new one. The patch grew in size as zygotes developed, eventually becoming a ring just prior to rhizoid outgrowth. The rhizoid emerged at the site of the F-actin ring and, following germination, the ring was located in the subapical zone of the elongating tip.

Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa

The predicted existence of cytoplasmic Ca2+ gradients during the photopolarization of the zygotes of the brown algae, Pelvetia and Fucus, has proved to be difficult to establish, and the downstream targets of the putative gradients are not known. We have used quantitative microinjection of the long excitation wavelength Ca2+ indicator, Calcium Crimson, and of antibodies against calmodulin to investigate these matters in the zygotes and early embryos of Pelvetia. We found that there is a window of cytoplasmic Calcium Crimson concentration that gives an adequate signal above autofluorescence yet allows normal development of the zygotes. As Calcium Crimson is not a ratiometric indicator, we injected other zygotes with a Ca2+-insensitive dye, rhodamine B, and imaged the cells at the same time that Calcium Crimson-injected cells were imaged. Ratios were calculated by dividing the averaged pixel values of Calcium Crimson images by the averaged pixel values of corresponding rhodamine B images. By this method, we observed the formation of a cytoplasmic Ca2+ gradient within one hour of the exposure of the cells to unilateral blue light during the photosensitive period. The region of high Ca2+ was localized to and predictive of the site of future rhizoid formation. We validated this somewhat indirect method by applying it to the growing rhizoid, where the existence of a tip-localized Ca2+ gradient is well established. The method clearly revealed the known gradient. The injection of ungerminated zygotes with antibodies made against Dictyostelium calmodulin inhibited germination, and this inhibition was abolished if the calmodulin antibodies were coinjected with an excess of purified maize calmodulin. Likewise, the growth of the rhizoids was inhibited by calmodulin antibody injections. The fungus-derived calmodulin antagonist, ophiobolin A, which has previously been shown to be a potent inhibitor of germination, also inhibited rhizoidal growth. Our results provide evidence that a cytoplasmic Ca2+ gradient is present during photopolarization and that calmodulin acts as a mediator of Ca2+ gradients throughout the early developmental processes of germination and rhizoidal growth in Pelvetia compressa.