No abstract

The increasing use of nanomaterials in healthcare and industrial products heightens the possibility of their ingestion by humans, other mammals, and fish. While toxicity of many nanomaterials has recently been studied, reports of non-lethal effects of nanomaterials remain ill-defined. This study investigates possible pathways by which nanoparticles, titanium dioxide (TiO(2)), could cross the epithelium layer by employing both toxicity and mechanistic studies. This study provides evidence that at 10 microg/mL and above, TiO(2) nanoparticles cross the epithelial lining of the intestinal model by transcytosis, albeit at low levels. TiO(2) was able to penetrate into and through the cells without disrupting junctional complexes, as measured by gamma-catenin. To monitor the epithelial integrity, transepithelial electrical resistance (TEER) was employed and determined low concentrations (10 or 100 microg/mL) of TiO(2) do not disrupt epithelial integrity. Live/dead analysis results did not show cell death after exposure to TiO(2). In addition, at 10 microg/mL (and above) TiO(2) nanoparticles begin alteration of both microvillar organization on the apical surface of the epithelium as well as induce a rise in intracellular-free calcium. The latter is a mechanism cells use to respond to extracellular stimuli and may be linked to the alteration of the apical microvilli. Although TiO(2) does not show cell death, the implication of other, non-lethal, effects could lead to undesired outcomes (i.e., disease, malnutrition, shortened life span, etc.).

Diethylene glycol distearate is used as a removable embedding medium to produce embeddment-free sections for transmission electron microscopy. The easily cut sections of this material float and form ribbons in a water-filled knife trough and exhibit interference colors that aid in the selection of sections of equal thickness . The images obtained with embeddment-free sections are compared with those from the more conventional epoxyembedded sections, and illustrate that embedding medium can obscure important biological structures, especially protein filament networks. The embeddment-free section methodology is well suited for morphological studies of cytoskeletal preparations obtained by extraction of cells with nonionic detergent in cytoskeletal stabilizing medium. The embeddment-free section also serves to bridge the very different images afforded by embedded sections and unembedded whole mounts .Transmission electron microscopic (TEM)" images of conventional thin sections are influenced by the presence of the embedding material to a degree not often appreciated . The embedding resin, especially epoxy, scatters electrons very much the way the embedded specimen does, rendering it nearly invisible, except where heavy-metal stains are bound (9) . The thin section is most effectively stained at its surface (I1, 18) and is thus essentially a two-dimensional slice of three-dimensional objects whose actual morphology can only be reconstructed with difficulty. Such images formed from exceedingly thin planes are excellent for visualizing many biological structures but are inappropriate for examining three-dimensional networks of protein filaments (5) such as those constituting the cytoskeletal framework (i.e., surface lamina and a protein filament network surrounding the chromatin-containing nucleus : reference 13) .An alternate but under utilized method of electron microscopy, the unembedded whole mount, provides an image 'Abbreviations used in this paper: DGD, diethylene glycol distearate ; MDCK, Madin-Darby canine kidney; nBA, n-butyl alcohol; TEM, transmission electron microscopy . directly from the specimen without resort to heavy-metal stains . In this procedure, the entire fixed, dehydrated, and critical-point dried cell is placed in the electron beam path in vacuo and biological material scatters electrons sufficiently to form very high contrast images. The important differences between stained, embedded sections and unstained whole mounts has been discussed in detail by Wolosewick and Porter (23,24) for unextracted, fixed (i.e., intact) cells .The embeddment-free whole mount has proven essential to the study of the cell architecture remaining after cells have been extracted with nonionic detergent. This procedure removes most lipids and soluble components leaving the cytoskeletal framework (for reviews see references 13 and 14) . This entity, revealed by detergent extraction, retains the configuration of the unextracted cell . Such preparations appear quite empty of structural elements when viewed in ...

Signaling events mediate many processes that act during embryogenesis to initiate the program of early development. Within the cell many of these changes are mediated through the activation or inactivation of kinases and phosphatases. Protein kinase C (PKC) is one kinase that has been shown to be involved in at least two developmental transitions during early development, fertilization and embryonic compaction. PKC is a family of kinases whose various isotypes have differing requirements for activation of the kinase that include the availability of calcium, diacylglycerol, and negatively charged phospholipids. The presence of more than one isotype in an egg or blastomere of the embryo would provide the possibility that different isotypes mediate distinct signaling pathways in the cells. To address this possibility the different isotypes of PKC were examined at the mRNA and protein levels during preimplantation development in the mouse. Our results demonstrate that seven isotypes of PKC are present during preimplantation development in mouse, some are of maternal origin and others appear after fertilization. Two isotypes have a stage-dependent nuclear localization. In addition, within each blastomere PKC isotypes occupy different subcellular locations in a stage-dependent fashion.

Bulk- and nano-scale titanium dioxide (TiO2) has found use in human food products for controlling color, texture, and moisture. Once ingested, and because of their small size, nano-scale TiO2 can interact with a number of epithelia that line the human gastrointestinal tract. One such epithelium responsible for nutrient absorption is the small intestine, whose constituent cells contain microvilli to increase the total surface area of the gut. Using a combination of scanning and transmission electron microscopy it was found that food grade TiO2 (E171 food additive coded) included ∼25% of the TiO2 as nanoparticles (NPs; <100 nm), and disrupted the normal organization of the microvilli as a consequence of TiO2 sedimentation. It was found that TiO2 isolated from the candy coating of chewing gum and a commercially available TiO2 food grade additive samples were of the anatase crystal structure. Exposure to food grade TiO2 additives, containing nanoparticles, at the lowest concentration tested within this experimental paradigm to date at 350 ng/mL (i.e., 100 ng/cm(2) cell surface area) resulted in disruption of the brush border. Through the use of two independent techniques to remove the effects of gravity, and subsequent TiO2 sedimentation, it was found that disruption of the microvilli was independent of sedimentation. These data indicate that food grade TiO2 exposure resulted in the loss of microvilli from the Caco-2BBe1 cell system due to a biological response, and not simply a physical artifact of in vitro exposure.

Elevation of intracellular free calcium causes egg activation by initiating a cascade of interacting signaling pathways that, in unison, act to remodel the cytoplasmic compartment and the nuclear compartment of the egg. We show here that calcium/calmodulin-dependent protein kinase II (CaM kinase II) is tightly associated with the meiotic spindle and that 5 min after egg activation there is a transient, tight association of calmodulin (colocalized with CaM kinase II) on the meiotic spindle. These correlative observations caused us to test whether activation of CaM kinase II mediated the chromosomal transit into an anaphase configuration. We demonstrate that calcium and calmodulin, at physiological levels, along with ATP were capable of driving the spindle (with its associated CaM kinase II) into an anaphase configuration in a permeabilized egg system. The transit into anaphase was dependent on the presence of both calcium and calmodulin and occurred normally when they were present at a ratio of 4 to 1. Peptide and pharmacologic inhibitors of CaM kinase II blocked the transit into anaphase, both in the permeabilized egg system and in living eggs (inhibitors of protein kinase C did not block the transit into anaphase). Using a biochemical approach we confirm that CaM kinase II increases in activity 5 min after egg activation and that a second increase occurs 45 min after activation at the approximate time that the contractile ring of the second polar body is constricting. This corresponds to the approximate time when calmodulin and CaM kinase II colocalize at several points in the activated egg including the region containing midzone microtubules. CaM kinase II appears localized on midzone microtubules as soon as they form and may have a role in specifying the position of the contractile ring of the second polar body.

No abstract

Abstract. Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.T HE program of fertilization in frog eggs is similar to that of sea urchin eggs (20, 49), fish eggs (24, 25), and mammalian eggs (17,40) in that it involves a wavelike increase in intracellular free calcium (7, 36), which precedes a wave of cortical granule exocytosis (26, 36). Cortical granule exocytosis in the frog Xenopus laevis is followed by contraction of the cortex and later, the formation of the first cleavage furrow (for a table of postfertilization events, see reference 55). Cortical granule exocytosis, cortical contraction, and cleavage furrow formation are the morphological hallmarks of frog egg activation, and provide convenient markers for use in the dissection of the activation process (21, 28).The discovery that inositol-l,4,5-trisphosphate (IPa),* an intermediate of the polyphosphoinositide pathway, triggered the intracellular wave of calcium and activated Xenopus eggs led to the suggestion that polyphosphoinositide turnover mediates the activation of Xenopus eggs (8, 44). Added support for this hypothesis is derived from studies of fertilization in sea urchins, mammals, and fish which indicate that IP3 formation triggers the wave of increase in intracellular free calcium (16,39,43,52,54). More recently, this hypothesis has been further strengthened by the finding that stimulation of receptors that trigger IP3 formation induces activation of Xenopus eggs (35).1. Abbreviations used in this paper: IP3, inositol-l,4,5-triphosphate; PD, phorbol 12,13-didecanoate; PDD, 4 ct-phorbol 12,13-didecanoate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; OAG, 1-olyeolyl-2-acetyl-sn-glycerol.IP3 is one of two intermediates produced by the activity of phospholipase C in the polyphosphoinositide pathway. This bifurcating signal pathway results in the formation of IP3 with the stoichiometric production of diacylglycerol (for reviews, see reference 4 and 42) and the latter activates...