Salisbury, Jeffery L.

Abstract Background Platelet dense granule (DG) deficiencies, including Hermansky-Pudlak syndrome, are relatively common and frequently under-diagnosed hereditary platelet hypofunctional disorders. Among various laboratory tests, platelet whole mount (WM) transmission electron microscopy (TEM) is considered the gold standard method for diagnosing DG deficiencies. Nevertheless, platelet DG TEM testing is still largely a research tool and has not been thoroughly validated and standardized as a clinical test. Furthermore, reference ranges (RR) of DG count/platelet (DGC) have not been adequately established. Aim Our goals were to validate and standardize platelet WM TEM testing and establish reference ranges for platelet DGC. Methods Based on previously established methods (Blood, 33:598-606), we first optimized and standardized various pre-analytical, analytical and post-analytical procedures including sample matrix, sample stability, platelet-rich plasma (PRP) preparation, platelet mounting procedure and DG counting criteria. Then whole blood samples in ACD-B tubes were collected from a total of 128 healthy donors (64 males and 64 females). Platelet DGC, percentage of platelets without any DG (empty platelets), platelet count (PC), mean platelet volume (MPV) and immature platelet fraction (IPF) were measured. In addition, platelet aggregometry analyses were performed on citrated PRP samples from the same donors. Standard statistical analyses were employed in this study. Results DGC and platelet ultra-structure were stable when whole blood samples were stored at room temperature (RT) for up to 4 days. Reproducible DGC and adequate preservation of platelet ultrastructure depended on an optimized mounting method. The DG counting criteria were developed based on previous publications and guidance from Dr. James G. White. Using the same WM TEM images and DG counting criteria, agreements among different technologists (n=5) improved from 60% to 95%. Finally, DGC reference range study using 128 healthy donor samples (age range 18, 72 years) and scoring 100-200 platelets from each donor showed that the DGC distribution of each donor sample was left-skewed with a median of 15% of empty platelets (range: 3, 40). The median DGC was 2.6 with a range of 1.0—5.1. Reference ranges established at the lower 95th percentile for DGC empty platelets is ≤ 31% (95% CI 28, 34) and for the mid-95th percentiles of DGCs between 1.5 and 4.0. DGCs were weakly associated with MPV and IPF (ρ between 0.22 and 0.28); and inversely associated with the percentage of empty platelets (ρ between -0.26 and -0.25). DGC showed no significant association with PC, platelet maximum aggregation or primary slope of aggregation, nor age or gender. Conclusion We validated platelet WM TEM procedures and for the first time established an adult reference range for platelet DGC. The results of association analyses implied that immature platelets may have slightly more DGs. Disclosures: No relevant conflicts of interest to declare.

Abstract Imagine a raft in a canal between point A and point B. On that raft is a visible (fluorescent) cargo. Also, attached to that raft is a motor that will propel the raft only from A to B (anterograde transport). When the raft gets to point B, another motor is attached that can propel the raft, and its cargo, and the anterograde motor, back to point A (retrograde transport). Within a cell, the canals are microtubLiles, and a lot is known about anterograde and retrograde transport in some systems, but these phenomena have not been directly observed in a living, intact animal. Until now, that is. In a pair of very interesting papers, the laboratory of Jonathan Scholey has shown us convincing micrographs of anterograde and retrograde transport in an important animal model.

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents γ-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. γ-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal γ-tubulin. Recruitment of maternal γ-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm “priming” induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

Abstract Carbon nanotubes represent a creative alternative for applications in medicine as therapeutic vectors, imaging and controlled release of active molecules. Although the low density and small size of carbon nanotubes makes respiratory exposures to workers likely during the production or use of commercial products, the potential genotoxicity of multi-walled carbon nanotubes has not been investigated. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In order to investigate whether genetic damage was unique to SWCNT, we examined mitotic spindle aberrations following dosing of cells to multi-walled carbon nanotubes (MWCNT) at concentrations anticipated in the workplace. Immortalized and primary respiratory epithelial cells were exposed to 0.024, 0.24, 2.4, and 24μg/cm2 carbon nanotubes. The minimal dose of MWCNT considered for this research was based on the permissible exposure limit for particles with an aerodynamic diameter of less than 5 microns (Occupation Safety and Health Administration). MWCNT induced a dose response of disrupted centrosomes, mitotic spindles and aneuploid chromosome number. The data further showed that monopolar mitotic spindles comprised 95% of the disrupted mitoses. The monopolar phenotype of the disrupted mitotic spindles is in sharp contrast to the multi-polar spindle of SWCNT-induced disruption. Three-dimensional reconstructions showed carbon nanotubes integrated with the microtubules, the DNA and within the centrosome structure. The lower doses did not cause cytotoxicity or apoptosis 24 hours after exposure; however, after 72 hours, significant cytotoxicity was observed in the MWCNT-exposed cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is a key event in the progression of cancer and is correlated with tumor stage. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant doses. These results suggest caution should be used in the handling and processing of carbon nanotubes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5464. doi:1538-7445.AM2012-5464

Abstract Lung cancers are often characterized by numerical chromosome instability. Hexavalent chromium (Cr(VI)) is a well-known human lung carcinogen; however, its carcinogenic mechanism remains unknown. Studies show that solubility plays a key role in its carcinogenicity, with the particulate, or insoluble, compounds being the most potent carcinogens. Epidemiological and whole animal studies pinpoint particulate zinc chromate as the most potent Cr(VI) carcinogen; however, few studies have investigated its carcinogenic mechanism. Therefore, the goal of this study was to determine if zinc chromate induces numerical chromosome instability in human lung cells. We found that exposure to zinc chromate for 24 h did not induce aneuploidy but longer exposures induced both concentration- and time-dependent increases in aneuploid metaphases. For example, exposure to 0.1, 0.15 and 0.2 ug/cm2 zinc chromate for 120 h induced 28, 40 and 44 percent aneuploid metaphases, respectively, with increases in hypodiploid, hyperdiploid and tetraploid metaphases. Consistent with the aneuploidy data, a 24 h exposure to zinc chromate did not induce centrosome amplification but more chronic exposures induced concentration- and time-dependent increases in centrosome amplification in both interphase and mitotic cells. Exposure to 0.1, 0.15 and 0.2 ug/cm2 zinc chromate for 120 h induced centrosome amplification in 13.9, 18 and 21.3 percent of interphase cells and 21, 33 and 46 percent of mitotic cells. In order to determine the mechanism of centrosome amplification, we investigated the number of centrioles in each centrosome and found that cells with amplified centrosomes exhibited centrosomes with normal centriole pairs and centrosomes with centriolar defects indicating multiple mechanisms are involved in zinc chromate-induced centrosome amplification. Lastly, we found that chronic exposure to zinc chromate induced a prolonged G2 arrest. All together, these data indicate that chronic exposure to zinc chromate induces numerical chromosome instability via centrosome amplification in human lung cells. This work was supported by NIEHS grant ES016893 (J.P.W.), cooperative agreement #EP-08-01 through the Maine Space Grant Consortium (J.P.W.), EPA GRO Fellowship MA-91685401 (A.L.H.) and the Maine Center for Toxicology and Environmental Health at the University of Southern Maine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3473.

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca 2+ -binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (∼300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.