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.

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 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) 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.

The stilbenoid tyrosine kinase inhibitor, G6, suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo.

Using structure-based virtual screening, we previously identified a novel stilbenoid inhibitor of Jak2 tyrosine kinase named G6. Here, we hypothesized that G6 suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. We found that G6 inhibited proliferation of the Jak2-V617F expressing human erythroleukemia (HEL) cell line by promoting marked cell cycle arrest and inducing apoptosis. The G6-dependent increase in apoptosis levels was concomitant with increased caspase 3/7 activity and cleavage of PARP. G6 also selectively inhibited phosphorylation of STAT5, a downstream signaling target of Jak2. Using a mouse model of Jak2-V617F-mediated hyperplasia, we found that G6 significantly decreased the percentage of blast cells in the peripheral blood, reduced splenomegaly, and corrected a pathologically low myeloid to erythroid ratio in the bone marrow by eliminating HEL cell engraftment in this tissue. In addition, drug efficacy correlated with the presence of G6 in the plasma, marrow, and spleen. Collectively, these data demonstrate that the stilbenoid compound, G6, suppresses Jak2-V617F-mediated aberrant cell growth. As such, G6 may be a potential therapeutic lead candidate against Jak2-mediated, human disease.

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-alpha-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-alpha-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 Pah(enu2) 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 pah(enu2) mouse model for phenylketonuria.

The Pah(enu2) 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 Pah(enu2) 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.

Cytologic diagnosis of Lawsonia intracellularis proliferative ileitis in a Japanese snow macaque (Macaca fuscata).

Diffuse ileal thickening and ileocecocolic lymphadenomegaly were observed during exploratory laparotomy in a 2-year-old male Japanese snow macaque (Macaca fuscata) that had flu-like signs and diarrhea. Cytologic examination of ileal biopsy imprints revealed many mature, mildly karyolytic neutrophils and fewer well-differentiated lymphocytes, eosinophils, macrophages, and plasma cells in a background containing amorphous, necrotic material. Tightly cohesive sheets of moderately pleomorphic epithelial cells also were seen. The cytologic diagnosis was chronic, active, mixed inflammation with atypical epithelial cells and necrosis. Histologically, the mucosal and crypt epithelium was moderately hyperplastic with a loss of goblet cells, increased mitoses, and frequent crypt abscesses. Within the lamina propria and extending into the submucosa was a marked neutrophilic infiltrate, with low numbers of lymphocytes, histiocytes, plasma cells, and eosinophils. The histologic diagnosis was chronic, diffuse, marked suppurative and lymphocytic ileitis. Warthin-Starry silver staining of the ileal biopsy and imprint specimens demonstrated numerous pleomorphic, curved bacilli consistent with Lawsonia intracellularis. Polymerase chain reaction (PCR) and immunohistochemistry confirmed the identity of the infectious agent. L intracellularis infection may be underdiagnosed because silver stain is required to visualize the organism with light microscopy and because the pathognomonic crypt hyperplasia may be complicated by secondary pathologic changes. Application of silver stain to cytologic specimens should be considered when distal intestinal lesions associated with hyperplastic epithelium, with or without inflammation, hemorrhage, or necrosis, are identified in animals with clinical signs of enteritis, especially in frequently affected species or in stressed or young animals.

Synovial fluid from an African spur-thighed tortoise (Geochelone sulcata).

A 4.5-year old, male African spur-thighed tortoise (Geochelone sulcata) was presented to the University of Florida Veterinary Teaching Hospital with a 2-week history of lethargy, anorexia, constipation, dyspnea, and coughing up fluid or vomiting. Laboratory results included an inflammatory leukogram and a marked increase in plasma uric acid concentration. Synovial fluid from multiple joints was thick, chalky white, and opaque, with a grainy consistency. Microscopically, the fluid contained numerous brown, needle-like crystals consistent with urates (gout). Gross necropsy findings and histopathology confirmed a diagnosis of systemic gout, with urate deposition, gout tophi, and underlying necrosis in multiple organs, including kidneys, lung, and liver. Dehydration with concurrent renal insufficiency may have impaired urate excretion and led to a build-up of urates in the blood and tissues of this tortoise. A high protein diet also may have contributed to the development of gout. Cytologic evaluation of synovial fluid can be used as a quick and definitive tool to diagnose gout in tortoises.