Removal of Toxoplasma gondii oocysts from sea water by eastern oysters (Crassostrea virginica).

Toxoplasma gondii infections have been reported in a number of marine mammals. Presently it is not known how these animals acquire T. gondii from their aquatic environment. The eastern oyster, Crassostrea virginica, has been shown to remove Cryptosporidiwn oocysts from seawater and a similar phenomenon may be occurring with T. gondii oocysts and marine invertebrates. The present study was done to determine if eastern oysters could remove and retain T. gondii oocysts from seawater. Oocysts of the VEG strain of T. gondii (1 x 10(6) oocysts) were placed in seawater (32 ppt NaCl) containing live eastern oysters. The infected seawater was removed one day postinoculation (PI) and replaced with fresh seawater. Selected oysters were removed at 1, 3 and 6 days PI. Hemolymph, gill washes, and oyster tissue were collected separately at each observation time. The oyster tissue was homogenized and all 3 samples fed separately to mice. Toxoplasma gondii positive mice were observed at each time period. The results indicate that T. gondii oocysts can be removed from seawater by eastern oysters and retain their infectivity. Contaminated raw oysters may serve as a source of T. gondii infection for marine mammals and humans.

Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium parvum viability.

Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti-Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT-8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT-8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 microg/ml produced a 97.8% reduction in infection.

NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism.

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.

Bundling of microtubules by motor and tail domains of a kinesin-like calmodulin-binding protein from Arabidopsis: regulation by Ca(2+)/Calmodulin.

Kinesin-like calmodulin-binding protein (KCBP), a novel kinesin-like protein from plants, is unique among kinesins and kinesin-like proteins in having a calmodulin-binding domain adjacent to its motor domain. KCBP localizes to mitotic microtubule (MT) arrays including the preprophase band, the spindle apparatus, and the phragmoplast, suggesting a role for KCBP in establishing these MT arrays by bundling MTs. To determine if KCBP bundles MTs, we expressed C-terminal motor and N-terminal tail domains of KCBP, and used the purified proteins in MT bundling assays. The 1.5 C protein with the motor and calmodulin-binding domains induced MT bundling. The 1.5 C-induced bundles were dissociated in the presence of Ca(2+)/calmodulin. Similar results were obtained with a 1.4 C protein, which lacks much of the coiled-coil region present in 1.5 C protein and does not form dimers. The N-terminal tail of KCBP, which contains an ATP-independent MT binding site, is also capable of bundling MTs. These results, together with the KCBP localization data, suggest the involvement of KCBP in establishing mitotic MT arrays during different stages of cell division and that Ca(2+)/calmodulin regulates the formation of these MT arrays.

N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain.

N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd motor-stalk protein MC1 (modified claret 1) was treated with varying concentrations of NEM (0-10 mM) and cosedimentation and ATPase assays were used to assess the effects of modification on MC1 interactions with MTs. In the cosedimentation assay, treatment with /=0.5 mM NEM induced aggregation of MC1 and resulted in sedimentation of the motor in the absence of MTs. NEM modification had no effect on the basal ATPase rate but produced a decrease in the MT-stimulated ATPase rate. Labeling of MC1 with [3H]NEM indicated that enhanced MT binding was associated with an average labeling of 1 Cys residue per MC1 polypeptide, while aggregation was associated with an average labeling of 2 Cys residues per MC1 polypeptide. Protein digestion, structural analysis, and mass spectrometry indicate that modification of Cys313 or Cys324 in the stalk domain is correlated with enhanced binding of MC1 to MTs. These results suggest that NEM enhances Ncd binding to MTs by disruption of neck and/or stalk function and demonstrate the importance of this region in motor function.