Crystal structure of the apo forms of psi 55 tRNA pseudouridine synthase from Mycobacterium tuberculosis: a hinge at the base of the catalytic cleft

The three-dimensional structure of the RNA-modifying enzyme, psi55 tRNA pseudouridine synthase from Mycobacterium tuberculosis, is reported. The 1.9-A resolution crystal structure reveals the enzyme, free of substrate, in two distinct conformations. The structure depicts an interesting mode of protein flexibility involving a hinged bending in the central beta-sheet of the catalytic module. Key parts of the active site cleft are also found to be disordered in the substrate-free form of the enzyme. The hinge bending appears to act as a clamp to position the substrate. Our structural data furthers the previously proposed mechanism of tRNA recognition. The present crystal structure emphasizes the significant role that protein dynamics must play in tRNA recognition, base flipping, and modification.     

5-oxo-6,8,11,14-eicosatetraenoic acid stimulates the release of the eosinophil survival factor granulocyte/macrophage colony-stimulating factor from monocytes

Allergic diseases such as asthma are characterized by tissue eosinophilia induced by the combined effects of chemoattractants and cytokines. Lipid mediators are a major class of endogenous chemoattractants, among which 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most potent for human eosinophils. In this study, we investigated the effects of 5-oxo-ETE on eosinophil survival by flow cytometry. We found that this compound could promote eosinophil survival in the presence of small numbers of contaminating monocytes, but not in their absence. The conditioned medium from monocytes treated for 24 h with 5-oxo-ETE also strongly promoted eosinophil survival, whereas the medium from vehicle-treated monocytes had no effect. An antibody against the granulocyte/macrophage colony-stimulating factor (GM-CSF) completely blocked the response of eosinophils to the conditioned medium from 5-oxo-ETE-treated monocytes, whereas an antibody against interleukin-5 had no effect. Furthermore, 5-oxo-ETE stimulated the release of GM-CSF from cultured monocytes in amounts compatible with eosinophil survival activity, with a maximal effect being observed after 24 h. This effect was concentration-dependent and could be observed at concentrations in the picomolar range. 5-Oxo-ETE and leukotriene B(4) had similar effects on GM-CSF release at low concentrations, but 5-oxo-ETE induced a much stronger response at concentrations of 10 nm or higher. This is the first report that 5-oxo-ETE can induce the release of any cytokine, suggesting that it could be an important mediator in allergic and other inflammatory diseases due both to its chemoattractant properties and to its potent effects on the synthesis of the survival factor GM-CSF.     

Formation and antiproliferative effect of prostaglandin E(3) from eicosapentaenoic acid in human lung cancer cells

We investigated the formation and pharmacology of prostaglandin E(3) (PGE(3)) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE(3.) The extracellular ratio of PGE(3) to PGE(2) increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE(3), respectively, than that produced by COX-1. Exposure of A549 cells to 1 microM PGE(3) inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE(3), however, had no effect. When A549 cells were exposed to EPA (25 microM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 microM). This effect appears to be associated with a 50% reduction of PGE(3) formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE(2), an increase in the level of PGE(3), and PGE(3)-mediated inhibition of tumor cell proliferation.     

GADD34 protein levels increase after transient ischemia in the cortex but not in the CA1 subfield: implications for post-ischemic recovery of protein synthesis in ischemia-resistant cells

Transient cerebral ischemia is a pathological process whereby an irreversible suppression of protein synthesis is believed to contribute to the extent of cell death in vulnerable neurons. Endoplasmic reticulum (ER) dysfunction has been identified as being responsible for ischemia-induced shut-down of translation. Recovery from ER dysfunction is facilitated by GADD34, a protein that dephosphorylates eukaryotic initiation factor (eIF)2alpha-P and thus reactivates protein synthesis. We investigated ischemia-induced changes in GADD34 levels in wild-type and Cu2+/Zn2+ SOD (SOD1) over-expressing rats. Transient global cerebral ischemia was induced by common carotid artery occlusion. Tissue samples were taken from the vulnerable hippocampal CA1 subfield and the resistant cerebral cortex of the right and left hemispheres for evaluation of changes in gadd34 mRNA and GADD34 protein levels. In wild-type animals, we found significantly lower GADD34 levels than in SOD1 transgenes but no differences in gadd34 mRNA levels, implying that superoxides regulate gadd34 translation. After ischemia, GADD34 protein levels were significantly increased in the cortex but not in the CA1 subfield, and these changes occurred earlier in SOD1 transgenic than in wild-type animals. The rise in gadd34 mRNA levels did not differ in the cortex and CA1 subfield, implying that gadd34 expression is regulated at the translational level.     

Identification of tyrosine hydroxylase as a physiological substrate for Cdk5

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.     

Induction of potent humoral and cell-mediated immune responses by attenuated vaccinia virus vectors with deleted serpin genes

Vaccinia virus (VV) has been effectively utilized as a live vaccine against smallpox as well as a vector for vaccine development and immunotherapy. Increasingly there is a need for a new generation of highly attenuated and efficacious VV vaccines, especially in light of the AIDS pandemic and the threat of global bioterrorism. We therefore developed recombinant VV (rVV) vaccines that are significantly attenuated and yet elicit potent humoral and cell-mediated immune responses. B13R (SPI-2) and B22R (SPI-1) are two VV immunomodulating genes with sequence homology to serine protease inhibitors (serpins) that possess antiapoptotic and anti-inflammatory properties. We constructed and characterized rVVs that have the B13R or B22R gene insertionally inactivated (vDeltaB13R and vDeltaB22R) and coexpress the vesicular stomatitis virus glycoprotein (v50DeltaB13R and v50DeltaB22R). Virulence studies with immunocompromised BALB/cBy nude mice indicated that B13R or B22R gene deletion decreases viral replication and significantly extends time of survival. Viral pathogenesis studies in immunocompetent CB6F(1) mice further demonstrated that B13R or B22R gene inactivation diminishes VV virulence, as measured by decreased levels of weight loss and limited viral spread. Finally, rVVs with B13R and B22R deleted elicited potent humoral, T-helper, and cytotoxic T-cell immune responses, revealing that the observed attenuation did not reduce immunogenicity. Therefore, inactivation of immunomodulating genes such as B13R or B22R represents a general method for enhancing the safety of rVV vaccines while maintaining a high level of immunogenicity. Such rVVs could serve as effective vectors for vaccine development and immunotherapy.     

Murine cytomegalovirus with a transposon insertional mutation at open reading frame m155 is deficient in growth and virulence in mice

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.     

TCDD-induced homologous recombination: the role of the Ah receptor versus oxidative DNA damage

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits numerous biological responses including carcinogenicity. The molecular mechanism by which TCDD exerts its tumorigenic effects is unclear, since it does not directly damage DNA. TCDD-initiated toxicity can be mediated by the aryl hydrocarbon receptor (AhR) pathway and/or via increased oxidative stress. DNA damage, including DNA oxidation, can induce DNA double-strand breaks, which can be repaired through homologous recombination. Excessive DNA double-strand breaks may promote aberrant DNA recombination, which can lead to detrimental genetic changes and ultimately to carcinogenesis. TCDD has been shown to induce homologous recombination but the molecular mechanism mediating these events are unknown. To investigate the role of the AhR and oxidative DNA damage in mediating TCDD-induced homologous recombination we used a Chinese hamster ovary (CHO) cell line containing a neo direct repeat recombination substrate (CHO 3-6). CHO 3-6 cells were exposed to TCDD (50, 500 or 1000 pM) in the presence or absence of an AhR antagonists (0.1 microM alpha-naphthoflavone (alpha-NF)) for 6 or 24 h and 2 weeks later homologous recombination frequencies were determined by counting the number of neo expressing, G418-resistant colonies per live cells plated. TCDD-initiated DNA oxidation was determined by measuring the formation of 8-hydroxy-2'-deoxyguanosine via HPLC and electrochemical detection. Exposure to 500 pM TCDD for 24 h significantly increased the frequency of homologous recombination. Southern blot analysis on G418-resistant colonies determined that TCDD induced both conservative gene conversion events and deletion events. DNA oxidation was not increased in cells exposed to TCDD for either 6 or 24 h. However, alpha-naphthoflavone exposure resulted in a significant decrease in TCDD-induced homologous recombination frequency. These results suggest that TCDD-initiated homologous recombination in CHO 3-6 cells is mediated by the AhR and not via increased oxidative stress.     

Human papillomavirus DNA and liquid-based cervical cytology cotesting in screening and follow-up patient groups

OBJECTIVE: To compare the use of human papillomavirus (HPV) DNA and cervical cytology cotesting in screening and follow-up of patients with previous cervical abnormalities and to assess the significance of a positive HPV DNA test result in re-screening of cytologically normal cases. STUDY DESIGN: Cellular samples collected in liquid-based fixative were used for both cervical cytology and HPV DNA testing. The cervical cytology slides were manually screened by cytotechnologists followed by rapid re-screening by pathologists. The HPV DNA tests were performed using hybrid capture test kits. Statistical analyses of cervical cytology results and HPV DNA tests for high- and low-risk HPV from both patient groups were carried out. RESULTS: The prevalence of HPV DNA-positive cases was higher in younger patients. There was a poor correlation between cervical cytology results and HPV DNA tests for the screening group (kappa = 0.23), but a fair to good correlation was obtained for the follow-up group (kappa = 0.51). The false negative fraction of cytology negative/HPV DNA positive cases (0.1317), as compared with cytology negative/HPV DNA negative cases (0.0056), was statistically significant (p = 0.000001). CONCLUSION: The prevalence of HPV DNA decreased with increasing age in both the screening and follow-up patient groups. Virus clearance was delayed in the follow-up group as compared with the screening group. There was a poor correlation between cervical cytology and HPV DNA tests in the screening group but a fair to good correlation in the follow-up patient group. Cotesting of HPV DNA and cervical cytology increases the sensitivity and decreases the false negative fraction, suggesting that cotesting could be used to increase the interval of screening.