James J. Faust

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. Mol. Reprod. Dev. 77: 95–104, 2010.

Survey of food-grade silica dioxide nanomaterial occurrence, characterization, human gut impacts and fate across its lifecycle

There is increasing recognition of the importance of transformations in nanomaterials across their lifecycle, yet few quantitative examples exist. We examined food-grade silicon dioxide (SiO2) nanomaterials from its source (bulk material providers), occurrence in food products, impacts on human gastrointestinal tract during consumption, and fate at wastewater treatment plants. Based upon XRD, XPS and TEM analysis, pure SiO2 present in multiple food-grade stock SiO2 exhibited consistent morphologies as agglomerates, ranging in size from below 100nm to >500nm, with all primary particle size in the range of 9-26nm and were most likely amorphous SiO2 based upon high resolution TEM. Ten of 14 targeted foods purchased in the USA contained SiO2 of the same morphology and size as the pristine bulk food-grade SiO2, at levels of 2 to 200mg Si per serving size. A dissolution study of pristine SiO2 showed up to 7% of the dissolution of the silica, but the un-dissolved SiO2 maintained the same morphology as the pristine SiO2. Across a realistic exposure range, pristine SiO2 exhibited adverse dose-response relationships on a cell model (microvilli) of the human gastro-intestinal tract, association onto microvilli and evidence that SiO2 lead to production of reactive oxygen species (ROS). We also observed accumulation of amorphous nano-SiO2 on bioflocs in tests using lab-cultured activated sludge and sewage sludges from a full-scale wastewater treatment plant (WWTP). Nano-scale SiO2 of the same size and morphology as pristine food-grade SiO2 was observed in raw sewage at a WWTP, but we identified non-agglomerated individual SiO2 particles with an average diameter of 21.5±4.7nm in treated effluent from the WWTP. This study demonstrates an approach to track nanomaterials from source-to-sink and establishes a baseline occurrence of nano-scale SiO2 in foods and WWTPs.

Engineered Nanoparticles Induced Brush Border Disruption in a Human Model of the Intestinal Epithelium

Nanoparticles hold great promise in cell biology and medicine due to the inherent physico-chemical properties when these materials are synthesized on the nanoscale. Moreover, their small size, and the ability to functionalize the outer nanoparticle surface makes them an ideal vector suited to traverse a number of physical barriers in the human body. While nanoparticles hold great promise for applications in cell biology and medicine, their downfall is the toxicity that accompanies exposure to biological systems. This chapter focuses on exposure via the oral route since nanomaterials are being engineered to act as carriers for drugs, contrast agents for specialized imaging techniques, as well as ingested pigments approved by regulatory agencies for human food products. After these nanomaterials are ingested they have the potential to interact with a number of biologically significant tissues, one of which is the epithelium of the small intestine. Within the small intestine exists enterocytes whose principal function is nutrient absorption. The absorptive process is aided by microvilli that act to increase the surface area of the epithelium. Dense arrays of microvilli, referred to as the brush border, have recently been shown to undergo disruption as a consequence of exposure to nanomaterials. This chapter aims to set the stage for detailed mechanistic studies at the cell biology level concerning this newly emerging nanotoxicity research paradigm, as the underlying structural characterization responsible for the existence of microvilli have been elucidated.

Commenting on the effects of surface treated- and non-surface treated TiO(2) in the Caco-2 cell model.

In a recent work published in Particle and Fibre Toxicology by Fisichella and coworkers investigating surface-modified TiO2 nanoparticle exposure in a model human intestinal epithelium (Caco-2), albeit degraded to mimic conditions in the gut and exposure to natural sunlight, purportedly resulted in no toxic effects. The authors (Fisichella et al.) claim to have confirmed the results of a 2010 report by Koeneman et al. However, the study by Koeneman and colleagues revealed significant effects of unmodified TiO2 nanoparticles. These contradicting data warrant further investigation into the possible effects of aluminum hydroxide, as these nanoparticles appear to have resulted in an abnormal apical surface in Caco-2 cells.This is a comment on http://www.particleandfibretoxicology.com/content/pdf/1743-8977-9-18.pdf.

A Facile Method for Separating and Enriching Nano and Submicron Particles from Titanium Dioxide Found in Food and Pharmaceutical Products.

Recent studies indicate the presence of nano-scale titanium dioxide (TiO2) as an additive in human foodstuffs, but a practical protocol to isolate and separate nano-fractions from soluble foodstuffs as a source of material remains elusive. As such, we developed a method for separating the nano and submicron fractions found in commercial-grade TiO2 (E171) and E171 extracted from soluble foodstuffs and pharmaceutical products (e.g., chewing gum, pain reliever, and allergy medicine). Primary particle analysis of commercial-grade E171 indicated that 54% of particles were nano-sized (i.e., < 100 nm). Isolation and primary particle analysis of five consumer goods intended to be ingested revealed differences in the percent of nano-sized particles from 32%‒58%. Separation and enrichment of nano- and submicron-sized particles from commercial-grade E171 and E171 isolated from foodstuffs and pharmaceuticals was accomplished using rate-zonal centrifugation. Commercial-grade E171 was separated into nano- and submicron-enriched fractions consisting of a nano:submicron fraction of approximately 0.45:1 and 3.2:1, respectively. E171 extracted from gum had nano:submicron fractions of 1.4:1 and 0.19:1 for nano- and submicron-enriched, respectively. We show a difference in particle adhesion to the cell surface, which was found to be dependent on particle size and epithelial orientation. Finally, we provide evidence that E171 particles are not immediately cytotoxic to the Caco-2 human intestinal epithelium model. These data suggest that this separation method is appropriate for studies interested in isolating the nano-sized particle fraction taken directly from consumer products, in order to study separately the effects of nano and submicron particles.

The Role of Integrins αMβ2 (Mac-1, CD11b/CD18) and αDβ2 (CD11d/CD18) in Macrophage Fusion

The subfamily of β2 integrins is implicated in macrophage fusion, a hallmark of chronic inflammation. Among β2 family members, integrin Mac-1 (αMβ2, CD11b/CD18) is abundantly expressed on monocyte/macrophages and mediates critical adhesive reactions of these cells. However, the role of Mac-1 in macrophage fusion leading to the formation of multinucleated giant cells remains unclear. Moreover, the role of integrin αDβ2 (CD11d/CD18), a receptor with recognition specificity overlapping that of Mac-1, is unknown. We found that multinucleated giant cells are formed in the inflamed mouse peritoneum during the resolution phase of inflammation, and their numbers were approximately twofold higher in wild-type mice than in Mac-1(-/-) mice. Analyses of isolated inflammatory peritoneal macrophages showed that IL-4-induced fusion of Mac-1-deficient cells was strongly reduced compared with wild-type counterparts. The examination of adhesive reactions known to be required for fusion showed that spreading, but not adhesion and migration, was reduced in Mac-1-deficient macrophages. Fusion of αDβ2-deficient macrophages was also significantly decreased, albeit to a smaller degree. Deficiency of intercellular adhesion molecule 1, a counter-receptor for Mac-1 and αDβ2, did not alter the fusion rate. The results indicate that both Mac-1 and αDβ2 support macrophage fusion with Mac-1 playing a dominant role and suggest that Mac-1 may mediate cell-cell interactions with a previously unrecognized counter-receptor(s).

Genistein supplementation prevents weight gain but promotes oxidative stress and inflammation in the vasculature of female obese ob/ob mice

Obesity, a state of chronic low-grade inflammation, is strongly associated with the development of hypertension and diabetes. Superoxide, a free radical elevated in obese individuals, promotes hypertension through scavenging the endogenous vasodilator nitric oxide. The hypothesis was a genistein-enriched diet would promote weight loss and reduce oxidative stress and inflammation in the vasculature of intact female ob/ob mice. Aortas and mesenteric arteries were isolated from female ob/ob mice fed genistein-free (0mg genistein/kg diet; n=6), standard chow (200-300mg genistein/kg diet; n=11) or genistein-enriched (600mg genistein/kg diet; n=9) diets for 4weeks. Sections of isolated vessels were labeled with the superoxide indicator dihydroethidium and fluorescence was measured by confocal microscopy. Protein expression of the inflammatory marker inducible nitric oxide synthase (iNOS) was measured in the perivascular adipose tissue (PVAT) surrounding each vessel and plasma concentrations of superoxide dismutase (SOD) were quantified. Genistein-enriched diet promoted less weight gain compared to animals fed standard chow (P=.008). Standard chow promoted increased superoxide in the aorta (P=.030) and mesenteric arteries (P=.024) compared to a diet devoid of genistein. At all tested concentrations, genistein significantly increased iNOS expression in mesenteric artery PVAT (vs. standard chow, P<.001; vs. genistein-enriched, P=.002) and tended to increase iNOS within the aortic PVAT (standard chow, P=.075) compared to the genistein-free group. Plasma SOD activity was significantly downregulated in genistein-enriched animals as compared to those fed a genistein-free diet (P=.028). In summary, although genistein prevents weight gain, it promotes vascular oxidative stress and inflammation in obese ovarian-intact female mice.

Multifunctional scaffolds in eggs: Sites for localization, signal transduction and meiotic spindle polarity

Molecular scaffolds in the mammalian egg are capable of tethering specific proteins involved in regulation of early development. Scaffolds can take the form of cytoskeletal elements, or involve proteins such as MARCKs or RACKSs during important cellular transitions in the egg. Moreover, with each cellular transition (i.e. germinal vesicle breakdown, meiosis I, meiosis II, etc) comes an extensive rearrangement of architectural elements within the cell. To accomplish this regulatory elements in signaling pathways should be in close molecular proximity to other discrete signaling pathways both to increase the speed of chemical reactions and to promote crosstalk. Crosstalk between signaling pathways is essential to modulate downstream effectors as one pathway can trigger activation/inhibition of another. It also is important to sequester or restrict access to various signaling enzymes for later use. These requirements create both morphological and biochemical heterogeneity, and likely necessitate the use of molecular scaffolds. This review examines the body of literature suggesting cytoskeletal elements serve to meet the aforementioned requirements in the mammalian egg.

Fabricating optical-quality glass surfaces to study macrophage fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

High glucose impairs acetylcholine-mediated vasodilation in isolated arteries from Mourning doves (Z. macroura).

Normal avian plasma glucose levels are 1.5-2 times greater than mammals of similar size. In mammals, hyperglycemia induces oxidative stress and impaired endothelium-dependent vasodilation. Prior work has shown that mourning doves have high levels of antioxidants and isolated vessels have low endogenous oxidative stress. Therefore, the hypothesis was that endothelium-dependent vasodilation of isolated avian arteries would not be impaired following acute exposure to high glucose. Isolated small resistance cranial tibial arteries (c. tibial) were cannulated and pressurized in a vessel chamber then incubated with either normal or high glucose (20mM vs. 30mM) for 1h at 41°C. Vessels were then pre-constricted to 50% of resting inner diameter with phenylephrine (PE) followed by increasing doses of acetylcholine (ACh; 10(-9) to 10(-5)M, 5min per step). Percent vasodilation was measured by tracking the inner diameter with edge-detection software. Contrary to our hypothesis, ACh-induced vasodilation was impaired with acute exposure to high glucose (p=0.013). The impairment was not related to increased osmolarity since vasodilation of arteries exposed to an equimolar combination of 20mM d-glucose and 10mM l-glucose was not different from controls (p=0.273). Rather, the impaired vasodilation was attributed to oxidative stress since superoxide levels were elevated 168±42% (p=0.02) and pre-exposure of arteries to the superoxide dismutase mimetic tiron (10mM) improved vasodilation (p<0.05). Therefore, isolated arteries from doves do not have endogenous mechanisms to prevent impaired vasodilation resulting from high glucose-mediated increases in oxidative stress.