William R. Huckle

Expression and Knockdown of Cellular Prion Protein (PrPC) in Differentiating Mouse Embryonic Stem Cells

The mammalian cellular prion protein (PrP(C)) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc)), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about pathogenic PrP conversion and its role in TSEs, the normal function of PrP(C) is poorly understood. Given the abundant expression of PrP(C) in the developing mammalian CNS and the spatial association with differentiated stages of neurogenesis, recently it has been proposed that PrP(C) participates in neural cell differentiation. In the present study, we investigated the role of PrP(C) in neural development during early embryogenesis. In bovine fetuses, PrP(C) was differentially expressed in the neuroepithelium, showing higher levels at the intermediate and marginal layers where more differentiated states of neurogenesis were located. We utilized differentiating mouse embryonic stem (ES) cells to test whether PrP(C) contributed to the process of neural differentiation during early embryogenesis. PrP(C) showed increasing levels of expression starting on Day 9 until Day 18 of ES cell differentiation. PrP(C) expression was negatively correlated with pluripotency marker Oct-4 confirming that ES cells had indeed differentiated. Induction of ES cells differentiation by retinoic acid (RA) resulted in up-regulation of PrP(C) at Day 20 and nestin at Day 12. PrP(C) expression was knocked down in PrP-targeted siRNA ES cells between Days 12 and 20. PrP(C) knockdown in ES cells resulted in nestin reduction at Days 16 and 20. Analysis of bovine fetuses suggests the participation of PrP(C) in neural cell differentiation during early embryogenesis. The positive association between PrP(C) and nestin expression provide evidence for the contribution of PrP(C) to ES cell differentiation into neural progenitor cells.

Developmental expression of the cellular prion protein (PrPC) in bovine embryos

The mammalian cellular prion protein (PrPC) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrPSc), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrPSc conversion and its role in TSEs, the normal function of PrPC has not been elucidated. In adult mammals, PrPC is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrPC is expressed during neurogenesis throughout development, and it has recently been proposed that PrPC participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell‐specific expression of PrPC during mammalian development, we examined PrPC expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre‐attachment embryos. PrPC was detected in the developing central and peripheral nervous systems in Day‐27, 32‐, and ‐39 embryos. PrPC was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrPC cell‐specific pattern of expression was detected in non‐nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrPC in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis. Mol. Reprod. Dev. 79:488–498, 2012.

Effects of Prolonged Tremor Due to Penitrem a in Mice

Adult mice were given subcutaneous injections of purified penitrem A (10 mg/kg) dissolved in corn oil. The mycotoxin was prepared by ether extraction and absorption chromatography; identity was established by absorption and mass spectra. Tremors were sustained for 72 hr following a single dose; readministration every 3 days was used to provide continuous trembling for 18 days. No lesions specifically attributed to penitrem A could be detected by histological examination of brains even after 18 days of trembling. Pharmacological agents affecting central nervous system neurotransmitters had some capacity to modify the effects of penitrem A. The results of those studies were such that a definitive conclusion regarding mechanisms of action of the toxin could not be determined.

Aqueous humor vascular endothelial growth factor in dogs: association with intraocular disease and the development of pre-iridal fibrovascular membrane.

OBJECTIVE To examine the concentrations of vascular endothelial growth factor (VEGF) in aqueous humor of dogs with intraocular disease and to evaluate the association of aqueous humor VEGF with pre-iridal fibrovascular membrane (PIFM) formation. PROCEDURES Two hundred and twenty six aqueous humor samples and 101 plasma samples were collected from 178 dogs with a variety of intraocular diseases (including cataract, primary glaucoma, uveitic glaucoma, aphakic/pseudophakic glaucoma, retinal detachment, lens luxation, and intraocular neoplasia), and aqueous humor was collected from 13 ophthalmically normal control dogs. Systemic disease status and administration of select medications were recorded. Aqueous and plasma VEGF was assayed via enzyme-linked immunosorbant assay validated for canine VEGF. Available histopathology samples were examined for the presence of PIFM. Where present, PIFMs were categorized as none, cellular, vascular or fibrous, and fibrovascular. Data were evaluated by mixed model anova, with application of Tukey-Kramer adjustment for multiple comparisons. RESULTS There was no association between aqueous humor and plasma VEGF levels. Compared with normal controls, aqueous humor VEGF was significantly elevated for all intraocular diseases (P < 0.05) except for primary and diabetic cataracts. Systemic disease and administered medications had no significant effect on aqueous humor VEGF concentration. Compared to dogs without PIFM, aqueous humor VEGF was significantly higher in dogs with fibrovascular PIFM (P = 0.001), but not cellular (P = 0.1704) or fibrous/vascular PIFM (P = 0.0667). CONCLUSIONS These findings confirm that VEGF is commonly elevated in aqueous humor of dogs with intraocular disease and likely plays a role in the causation or progression of a variety of intraocular disorders, including the development of PIFM.

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ–mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b+ cells suggested that IFN-γ production by microglia, which is the only CD11b+ cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell–mediated immune responses to control cerebral infection with T. gondii.

Expression of vascular endothelial growth factor receptor‐1 and ‐2 in normal and diseased canine eyes

OBJECTIVE   To immunohistochemically evaluate expression of vascular endothelial growth factor receptor-1 (VEGFR1) and -2 (VEGFR2) in ocular tissue of healthy dogs and dogs affected with primary glaucoma, uveitic glaucoma, and intraocular neoplasia. SAMPLE POPULATION   Enucleated globes from five dogs with primary glaucoma, five dogs with uveitic glaucoma, six dogs with intraocular neoplasms and three ophthalmically normal control dogs. PROCEDURE   Ocular tissues were obtained from enucleated globes of clinical cases or immediately following euthanasia for control dogs. Tissue sections were stained immunohistochemically for VEGFR1 and VEGFR2 via standard techniques and vascular tissue was qualitatively evaluated. Vascular endothelial VEGFR1 and VEGFR2 expression patterns are reported for normal and diseased ocular tissues. In addition, VEGFR1 and VEGFR2 expression patterns are reported for all normal ocular tissues. RESULTS   A constitutive expression pattern was detected for VEGFR1 by ocular vascular endothelial cells as well as nonvascular cells in the cornea, uvea, lens, and retina. VEGFR2 demonstrated limited expression in normal ocular tissue, but was widely expressed in vascular endothelium of diseased eyes, particularly in pre-iridal fibrovascular membranes. CONCLUSIONS   The results of this study suggest a role for VEGF receptors in both physiologic and pathologic angiogenesis in canine ocular tissue. Manipulation of this pathway may be a rational consideration for therapeutic intervention in canine ocular disease exhibiting pathologic neovascularization.

Increase in glucuronide conjugation of aflatoxin P1 after pretreatment with microsomal enzyme inducers

Microsomes prepared from livers of chickens given enzyme inducers had increased capability to convert aflatoxin P1 to its glucuronide conjugate. This capability was 190% +/- 19 and 184% +/- 13 of control values (mean +/- S.E., N = 4) 96 h after intraperitoneal administration of 80 mg/kg beta-naphthoflavone or 3-methylcholanthrene, respectively. Glucuronidation of aflatoxin P1 was also increased 15 days after 500 mg/kg i.p. of a polychlorinated biphenyl mixture (to 471% +/- 111). Fifteen days on a low protein diet (containing 54% of normal levels) did not alter aflatoxin glucuronidation. Increased glucuronidation after administration of inducers was due to increased specific activity of the microsomal enzymes.

Cellular modes of adaptation to environmental changes

Eukaryotic cells are remarkably adaptable entities. Whether embedded in solid tissues or freely suspended in blood or other fluids, cells principally exist in an aqueous environment but maintain a hydrophobic barrier, the plasma membrane, across which changes in the environment are detected. Utilizing specialized macromolecular components, cells can sense changes in temperature, hydrostatic pressure, oxygen tension, shear, shape, osmolarity, pH, electrical potential, electromagnetic radiation, and the concentrations of specific chemical compounds. Modes of response are equally varied, ranging from rapid secretion of stored substances to irreversible functional differentiation to self-destruction. Recent research has elucidated many of the enzymatic and genetic programs that accomplish these adaptations and suggests novel targets for therapeutic intervention.