Human intestinal epithelial cells exhibit a cellular response indicating a potential toxicity upon exposure to hematite nanoparticles.

This study examined the effects of different-sized nanoparticles on potential cytotoxicity in intestinal epithelia. Three sizes of hematite nanoparticles were used for the study at a 10 ppm concentration: 17, 53, and, 100 nm. Results indicate that, of the hematite nanoparticles tested, 17 nm was more toxic to the epithelial integrity than 53 or 100 nm. In addition, the epithelial integrity was affected by disruption of epithelial structures such as apical microvilli, and by disruption of the cell-cell junctions leading to reduction in transepithelial electrical resistance measurements (TEER). The drop in TEER was caused by disruption of the adhering junctions not by cell death, as determined by immunocytochemistry, and by using a cell viability assay. Epithelial integrity was also affected at the molecular level as shown by differential expression of genes related to cell junction maintenance, which was assessed by microarray analysis. In conclusion, the 17- and 100-nm hematite nanoparticles caused significant structural changes in the epithelium but not the 53 nm nanoparticles. Also, different-sized hematite nanoparticles each had different effects both at the cellular level and genetic level.

Involvement of PKCζ and GSK3ß in the stability of the metaphase spindle.

In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3ß (GSK3ß) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3ß are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3ß was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3ß. Second, the involvement of inactive GSK3ß in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3ß. Phospho-PKCζ is in close molecular proximity to GSK3ß, whereas the other isoforms of PKC such as pPKCßII, pPKCγ, pPKCµ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3ß may be a substrate of PKCζ.

Adsorption of hematite nanoparticles onto Caco-2 cells and the cellular impairments: effect of particle size.

The increasing applications of engineered nanomaterials nowadays have elevated the potential of human exposure through various routes including inhalation, skin penetration and digestion. To date there is scarce information on a quantitative description of the interactions between nanoparticles (NPs) and cell surfaces and the detrimental effects from the exposure. The purpose of this work was to study in vitro exposure of Caco-2 cells to hematite (alpha-Fe(2)O(3)) NPs and to determine the particle size effects on the adsorption behaviors. Cellular impairment was also investigated and compared. Hematite NPs were synthesized as part of this study with a discrete size distribution and uniform morphology examined by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Caco-2 cells were cultured as a model epithelium to mirror human intestinal cells and used to evaluate the impacts of the exposure to NPs by measuring transepithelial electrical resistance (TEER). Cell surface disruption, localization and translocation of NPs through the cells were analyzed with immunocytochemical staining and confocal microscopy. Results showed that hematite NPs had mean diameters of 26, 53, 76 and 98 nm and were positively charged with minor aggregation in the buffer solution. Adsorption of the four sizes of NPs on cells reached equilibrium within approximately 5 min but adsorption kinetics were found to be size-dependent. The adsorption rates expressed as mg m(-2) min(-1) were greater for large NPs (76 and 98 nm) than those for small NPs (26 and 53 nm). However, adsorption rates, expressed in units of m(-2) min(-1), were much greater for small NPs than large ones. After the adsorption equilibrium was reached, the adsorbed mass of NPs on a unit area of cells was calculated and showed no significant size dependence. Longer exposure time (>3 h) induced adverse cellular effects as indicated by the drop in TEER compared to the control cells without the exposure to NPs. NPs initially triggered a dynamic reorganization and detachment of microvilli structures on Caco-2 cell surfaces. Following this impact, the drop in TEER occurred more significantly, particularly for the exposure to 26 nm NPs, which was consistent with the observations with confocal microscopy that the junctions were more severely disrupted by 26 nm NPs than other sizes. In conclusion, this paper demonstrates the interactions at the ultrastructural level from initial surface adsorption of NPs upon cells, to the subsequent microvilli reorganization, membrane penetration and the disruption of adherens junction and provides the fundamental information on size effects on NP behavior which is often poorly addressed for in vitro cytotoxicity studies of NPs.

Involvement of the PKC family in regulation of early development.

Protein kinase C (PKC) isotypes have been implicated in a number of key steps during gametogenesis, fertilization, and early development. The 11-member family of PKC isotypes, many with different cofactor requirements for activation, can provide for differential activation of the specific kinases. In addition the enrichment of particular PKC isotypes to unique locations within gametes, zygotes, and early embryos likely promotes specific substrate interactions. Evidence exists to indicate involvement of PKC isotypes during sperm capacitation and the acrosome reaction, during resumption of meiosis in the oocytes, regulating the spindle organization in meiosis I and II, at fertilization, in the pronuclei, in the mitotically dividing blastomeres of the embryo, and at the plasma membranes of blastomeres at the time of embryonic compaction. Evidence also exists for crosstalk with other signaling pathways and one or more isotypes of PKC appear to be active at each major developmental transition.

Drosophila doubletime mutations which either shorten or lengthen the period of circadian rhythms decrease the protein kinase activity of casein kinase I.

In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt(S)) and long-period (dbt(L)) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt(S) and dbt(L) mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT(S), and DBT(L) proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt(S) and dbt(L) mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKIdelta expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt(S) and dbt(L) mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.