No abstract

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.

The resounding success of a new immunosuppressive regimen known as the Edmonton protocol demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. However, under the Edmonton protocol, a single donor does not provide enough islets to attain the insulin independence of a transplant recipient. This limitation is mainly caused by islet apoptosis triggered during isolation. In this study, we describe a highly efficient system of transiently transferring antiapoptotic proteins into pancreatic islets, thus opening an exciting new therapeutic opportunity to improve the viability of transplantable islets. We fused ␤-galactosidase to the 11-amino acid residues that constitute the protein transduction domain (PTD) of the HIV/TAT protein and transduced pancreatic islets ex vivo with this fusion protein in a dose-dependent manner with >80% efficiency. We observed that transduction of the anti-apoptotic proteins Bcl-X L and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-␣ in a pancreatic ␤-cell line, indicating that TAT/PTD anti-apoptotic proteins retained their biological activity. Finally, we demonstrated that TAT-fusion proteins did not affect the insulin secretion capability of islets, as determined by glucose static incubation and by reversion of hyperglycemia in diabetic immunodeficient 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, VV⌬K3L, or VV⌬E3L. 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. VV⌬E3L 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 VV⌬E3L, with a distinct pathology. VV⌬K3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VV⌬K3L 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.Small-animal (mouse) models to study poxvirus pathogenesis have been employed for decades and have provided invaluable insight into host-virus interactions and mechanistic insight as to how individual viral genes influence the host response. Infection of mice by various routes continues to be productive for study of a number of orthopoxviruses, including vaccinia virus (VV), ectromelia virus, cowpox virus, and rabbitpox virus (23, 24, 44, 45, 48-50, 67, 75, 77). The general purpose of these infected mouse studies is to provide surrogate models for human smallpox virus infection and host response to evaluate antiviral drug efficacy and the effectiveness of various smallpox vaccines. Advantages of mouse models include their relatively low cost, small size of the animals, and the fact that there are many mouse mutant strains available for genes that control responses to infection.A number of routes for the initiation of poxvirus infections in mice have been studied, including intranasal (i.n.), intratracheal (i.t.), and intradermal (i.d.) (ear pinnae, footpad) routes and scarification. Other routes, such as the intracranial (i.c.) or intraperitoneal (i.p.) route, are rarely used today because they do not effectively reproduce "natural" infection conditions. Arguably, the most relevant routes of infection in terms of human disease are the i.n. and i.t. routes, because smallpox is transmitted via th...

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.

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.

Pulmonary arterial hypertension (PAH) is a life-threatening disease that results in right ventricular failure. 5- 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 Ͻ 0.05) improved right ventricular ejection fraction. PRX-08066 significantly reduced peak PA pressure at 50 and 100 mg/kg (P Ͻ 0.05 and Ͻ 0.01, respectively) compared with MCT control animals. PRX-08066 therapy also significantly reduced right ventricle (RV)/body weight and RV/left ventricle ϩ septum (P Ͻ 0.01 and Ͻ 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 Ͻ 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. Pulmonary arterial hypertension (PAH)is an elevation in pulmonary vascular resistance caused by vasoconstriction and pulmonary vascular remodeling, resulting in increased pulmonary arterial pressure. Idiopathic PAH has no known underlying cause. PAH can also develop as a consequence of coexisting disease, such as chronic obstructive pulmonary disease, hypoxia, portal hypertension, or HIV infection (Archer and Rich, 2000;Li et al., 2006). PAH is usually progressive and invariably fatal. The median survival without treatment in adult patients with PAH was 2.8 years after diagnosis, and in children the median survival was only 10 months (D'Alonzo et al., 1991). Recent advances in PAH therapies, specifically epoprostenoltreated patients, have resulted in the median survival reaching more than 6 years in adults (Barst et al., 1994) and significant improvement in survival for children with reported rates at 1, 5, and 10 years being 94, 81, and 61%, respectively (Yung et al., 2004). Although survival rates have improved with new therapeutic modalities, the prognosis is still poor and development of more effective therapies is clearly needed.Serotonin or 5-hydroxytryptamine (5-HT) was recognized for playing a role in the development of PAH because a relationship between PAH patients and diet pills such as aminorex, dexfenfluramine (DF), and fenfluramine was observed (Kew, 1970; ...

We have mapped and sequenced the epsilon globin gene and seven surrounding Alu repeat sequences in the orangutan beta globin gene cluster and have compared these and other orangutan sequences to orthologously related human sequences. Noncoding flanking and intron sequences, synonymous sites of alpha, gamma, and epsilon globin coding regions, and Alu sequences in human and orangutan diverge by 3.2%, 2.7%, and 3.7%, respectively. These values compare to 3.6% from DNA hybridizations and 3.4% from the psi eta globin gene region. If as suggested by fossil evidence and "molecular clock" calculations, human and orangutan lineages diverged about 10-15 MYA, the rate of noncoding DNA evolution in the two species is 1.0-1.5 X 10(-9) substitutions per site per year. We found no evidence for either the addition or deletion of Alu sequences from the beta globin gene cluster nor is there any evidence for recent concerted evolution among the Alu sequences examined. Both phylogenetic and phenetic distance analyses suggest that Alu sequences within the alpha and beta globin gene clusters arose close to the time of simian and prosimian primate divergence (about 50-60 MYA). We conclude that Alu sequences have been evolving at the rate typical of noncoding DNA for the majority of primate history.