Jennifer E. Embury

MTF-1-mediated repression of the zinc transporter Zip10 is alleviated by zinc restriction.

The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Additionally, Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP analysis, binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by physically blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy.

Roles of Vaccinia Virus Genes E3L and K3L and Host Genes PKR and RNase L during Intratracheal Infection of C57BL/6 Mice

ABSTRACT The importance of the 2′-5′ oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L−/− PKR−/− mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L−/− mice showed no disease, whereas 20% of the PKR−/− mice succumbed at a dose of 108 PFU. Lethal disease was routinely observed in RNase L−/− PKR−/− mice inoculated with 108 PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.

PRX-08066, a Novel 5-Hydroxytryptamine Receptor 2B Antagonist, Reduces Monocrotaline-Induced Pulmonary Arterial Hypertension and Right Ventricular Hypertrophy in Rats

Pulmonary arterial hypertension (PAH) is a life-threatening disease that results in right ventricular failure. 5-((4-(6-Chlorothieno[2,3-d]pyrimidin-4-ylamino)piperidin-1-yl)methyl)-2-fluorobenzonitrile monofumarate (PRX-08066) is a selective 5-hydroxytryptamine receptor 2B (5-HT2BR) antagonist that causes selective vasodilation of pulmonary arteries. In the current study, the effects of PRX-08066 were assessed by using the monocrotaline (MCT)-induced PAH rat model. Male rats received 40 mg/kg MCT or phosphate-buffered saline and were treated orally twice a day with vehicle or 50 or 100 mg/kg PRX-08066 for 5 weeks. Pulmonary and cardiac functions were evaluated by hemodynamics, heart weight, magnetic resonance imaging (MRI), pulmonary artery (PA) morphology, and histology. Cardiac MRI demonstrated that PRX-08066 (100 mg/kg) significantly (P < 0.05) improved right ventricular ejection fraction. PRX-08066 significantly reduced peak PA pressure at 50 and 100 mg/kg (P < 0.05 and < 0.01, respectively) compared with MCT control animals. PRX-08066 therapy also significantly reduced right ventricle (RV)/body weight and RV/left ventricle + septum (P < 0.01 and < 0.001, respectively) compared with MCT-treated animals. Morphometric assessment of pulmonary arterioles revealed a significant reduction in medial wall thickening and lumen occlusion associated with both doses of PRX-08066 (P < 0.01). The 5-HT2BR antagonist PRX-08066 significantly attenuated the elevation in PA pressure and RV hypertrophy and maintained cardiac function. Pulmonary vascular remodeling was also diminished compared with MCT control rats. PRX-08066 prevents the severity of PAH in the MCT rat model.

Impact of Pancreatic Cold Preservation on Rat Islet Recovery and Function

Background. Islet transplantation success depends on the number and quality of islets transplanted. This study aimed at exploring the molecular mechanisms associated with cold pancreas preservation and their impact on islet cell survival and function. Methods. Rat pancreata were stored in cold University of Wisconsin preservation solution for short (3 hr; control) or long (18 hr) cold ischemia times (CIT). Results. Pancreata exposed to long CIT yielded lower islet numbers and showed reduced cellular viability; isolated islets displayed higher levels of phosphorylated stress-activated protein kinase (c-jun N-terminal Kinase and Mitogen-Activated Protein Kinase-p38), and chemokine (C-C) ligand-3, and lower levels of vascular endothelial growth factor, interleukins (IL)-9 and IL-10. Islets obtained from long-CIT pancreata were functionally impaired after transplantation. Differential proteomic expression in pancreatic tissue after CIT included increased eukaryotic translation elongation factor-1-α-1 (apoptosis related) and reduced Clade-B (serine protease inhibitor). Conclusions. Our study indicates that cold ischemia stimulates inflammatory pathways (chemokine (c-c)ligand-3, phosphorylation of c-jun N-terminal Kinase and mitogen-activated protein kinase-p38, and eukaryotic translation elongation factor-1-α-1) and decreases repair/cytoprotective pathways (IL-10, vascular endothelial growth factor, and Clade-B), all of which may negatively affect the quality and mass of islets obtained from a donor pancreas.

PKU is a reversible neurodegenerative process within the nigrostriatum that begins as early as 4 weeks of age in Pahenu2 mice

Phenylketonuria (PKU) is a common genetic disorder in humans that arises from deficient activity of phenylalanine hydroxylase (PAH), which catalyzes the conversion of phenylalanine to tyrosine. There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination. The neuropathogenesis of the disease has not been completely elucidated, however, oxidative stress is considered to be a key feature of the disease process. Hyperphenylalanemia also adversely affects monoaminergic metabolism in the brain. For this reason we chose to evaluate the nigrostriatum of Pah(enu2) mice, to determine if alterations of monoamine metabolism resulted in morphologic nigrostriatal pathology. Furthermore, we believe that recent developments in adeno-associated virus (AAV)-based vectors have greatly increased the potential for long-term gene therapy and may be a viable alternative to dietary treatment for this metabolic disorder. In this study we identified neurodegenerative changes with regenerative responses in the nigrostriatum of Pah(enu2) mice that are consistent with oxidative injury and occurred as early as 4 weeks of age. These neuropathologic changes were reversed following portal vein delivery of a recombinant adeno-associated virus-mouse phenylalanine hydroxylase-woodchuck hepatitis virus post-transcriptional response element (rAAV-mPAH-WPRE) vector to Pah(enu2) mice and corresponded to rapid reduction of serum Phe levels.

Pathologic and Immunohistochemical Findings in Hypothalamic and Mesencephalic Regions in the Pahenu2 Mouse Model for Phenylketonuria

The Pahenu2 mouse, created through ethylnitrosurea mutagenesis, is a model for phenylketonuria. These mice have elevated serum phenylalanine levels, hypopigmentation, and behavior and movement abnormalities, and female mice exhibit a maternal phenylketonuria syndrome. We evaluated the brains of adult and juvenile Pahenu2 mice for consistent, demonstrable lesions to elucidate various neuropathologic processes and to assess the efficacy of various treatment modalities such as AAV-mediated gene therapy. One aspect of the disease may involve the effect of hyperphenylalanemia on catecholamine function. High levels of phenylalanine inhibit enzymes that are important in the conversion of tyrosine and tryptophan to their respective neurotransmitter derivatives, including dopamine. Therefore, assessment of dopaminergic regions was of interest in this study. Histologic evaluation of juvenile and adult brains revealed an increased cellular density as early as 4 wk of age in the middle to posterior hypothalamus and substantia nigra. The infiltrating cells showed immunoreactivity for CD11b and had morphologic characteristics of macrophages. There was marked expression of inducible nitric oxide synthase in these dopaminergic regions that co-localized to CD11b-positive cells. The CD11b-positive cells and increased inducible nitric oxide synthase expression in these regions may function in a neuroregulatory manner to compensate for alterations in dopamine metabolism.

Effects of Pancreas Cold Ischemia on Islet Function and Quality

We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .01). Islet cell viability after isolation was significantly reduced in the Long-CIT group (P < .05). Reversal of diabetes following transplantation of suboptimal islet grafts occurred earlier in the Short-CIT group than the Long-CIT. All animals in the Short-CIT group and 80% in the Long-CIT group achieved euglycemia. Freshly isolated islets showed a significant increase of JNK and p38 (P < .05) phosphorylation in Long-CIT compared with Short-CIT. Histopathological assessment of the pancreas showed a significantly higher injury score. Proteomic analysis of pancreatic tissue led to identification of 5 proteins consistently differentially expressed between Short-CIT and Long-CIT. Better understanding of the molecular pathways involved in this phenomenon will be of assistance in defining targeted interventions to improve organ use in the clinical arena.

214. Dominant-Negative Interference in the Pahenu2 Mouse: Modified Forms of PAH

Top of pageAbstract The common human genetic disorder Phenylketonuria (PKU) is primarily caused by defects in the enzyme phenylalanine hydroxylase (PAH). An animal model for PKU, the BTBR Pahenu2 mouse, has a missense mutation (F263S) that inactivates PAH; the mouse exhibits classic PKU, with elevated blood Phe levels, cognitive deficiencies, and maternal PKU syndrome. We have corrected both the serum Phe levels and maternal PKU in Pahenu2 mice using recombinant AAV vectors containing the mouse PAH gene. Although successful, unusually high vector doses were needed to achieve normal serum Phe levels. More critically, there was a gradual loss of therapeutic effect 20-24 weeks after vector delivery in many animals, particularly females. This was accompanied by changes in liver morphology suggestive of increased metabolic activity. Pahenu2 mice express reduced levels of a mutant PAH protein. The normal enzyme is a tetramer and we have reported evidence fordominant-negative interactions between endogenous mutant subunits and vector-introduced functional subunits. Possible approaches to combating a dominant-negative interaction include the production of ribozymes targeting the endogenous mutant mRNA and expression of modified forms of PAH protein that do not form tetramers. We have shown that a serotype 2 AAV vector expressing both a ribozyme directed against the mouse PAH mRNA and a ribozyme-resistant form of mPAH (mPAH-Hd) were effective at reducing serum Phe levels. We now show that modified forms of the PAH enzyme can, in certain cases, reduce dominant-negative interference. A vector expressing an obligate dimer form of mPAH was still subject to negative interference, indicating this inhibition occurs at the level of the monomer-monomer interaction. We will present in vitro and in vivo data examining the effectiveness of both a monomeric PAH enzyme as well as chimeric enzymes expressing a mouse PAH catalytic core linked to the rat tyrosine hydroxylase tetramerization domain in reducing dominant-negative interactions with missense subunits.

423. Increased Incidence of Hepatocellular Change and Neoplastic Disease in AAV-WPRE Treated Pahenu2 Mice

Phenylketonuria (PKU) is among the most common human birth defects occurring in approximately 1 in 16,000 births in the United States. The disease is caused by a single gene defect in the enzyme phenylalanine hydroxylase (PAH) which results in elevated serum phenylalanine (Phe) levels. Our laboratory is involved in the investigation of AAV applications for the treatment of this disease in a mouse model for PKU.

879. Morphologic and Immunohistochemical Evaluation of Dopaminergic Regions of Pahenu2 Mouse Brains Following Adminstration of rAAV-mPAH-WPRE Vector

Phenylketonuria (PKU) is one of the most common single gene disorders in man. The metabolic defect arises from a deficiency of phenylalanine hydroxylase (PAH). There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination; these functions are associated with various dopaminergic tracts.