Spermcasting of spermatozeugmata by the bivalves Nutricola confusa andN. tantilla

Abstract. The dynamics and consequences of the varied reproductive modes of marine invertebrates is a rich and vibrant field of inquiry for ecological and evolutionary studies. One mode of reproduction that is not as well-studied as others is "spermcasting" or "spermcast mating," when males broadcast sperm and females retain eggs and brood developing embryonic stages. This type of reproduction occurs in two small (maximum adult shell length~5-6 mm) venerid bivalves, Nutricola confusa and N. tantilla, that live in protected bays of the temperate eastern Pacific. Females of these species brood developing embryos in chambers formed by the inner demibranchs, and release fully formed juveniles. We discovered that upon exposing clams to fluvoxamine, a selective serotonin reuptake inhibitor, males release spermatozeugmata, clusters of sperm cells attached by their heads to a central core. Spermatozoa of Nutricola have unusually long, needle-shaped heads that are approximately one quarter of the total length of the cell. These heads are curled and "packaged" into the hemispherical-shaped cores of spermatozeugmata. The cores are about one-third as long as the heads, and the tails protrude out of the opposite side of the cap of the core. The spermatozeugmata display two different swimming patterns, one where the tails beat in synchrony, and the other where they do not. The size of the cores is not significantly different in the two species, but spermatozeugmata of N. tantilla have significantly longer and wider tails than those of N. confusa. Advantages to spermcasting spermatozeugmata instead of individual spermatozoa may include enhanced dispersal and increased probabilities of fertilization. One consequence of spermatozeugmata (rather than individual spermatozoa) entering female brood chambers might be lowering of the effective population size. For species like these, which lack pelagic larvae, spermatozeugmata could increase dispersal and gene flow

Mechanism of scrapie prion precipitation with phosphotungstate anions.

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes to give a lacunary [PW11O39](7-) (PW11) complex and a single orthotungstate anion [WO4](2-) (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrP(Sc) from brain homogenates of scrapie prion-infected mice. The amount of PrP(Sc) isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrP(Sc) precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP(Sc) were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP(Sc) in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP(Sc) aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP(Sc). We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP(Sc) by PTA from tissue and might be optimized further for the detection of prions

Mechanism of Scrapie Prion Precipitation with Phosphotungstate Anions

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP^Sc) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP^Sc under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW₁₂O₄₀]^3– ion decomposes to give a lacunary [PW₁₁O₃₉]^7– (PW₁₁) complex and a single orthotungstate anion [WO₄]^2– (WO₄). To measure the efficacy of each component of PTA, increasing concentrations of PW₁₁, WO₄, and mixtures thereof were used to precipitate PrP^Sc from brain homogenates of scrapie prion-infected mice. The amount of PrP^Sc isolated, quantified by ELISA and immunoblotting, revealed that both PW₁₁ and WO₄ contribute to PrP^Sc precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP^Sc were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP^Sc in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP^Sc aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP^Sc. We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP^Sc by PTA from tissue and might be optimized further for the detection of prions