DNA-binding behavior of ruthenium(II) complexes containing both group 15 donors and 2,2':6',2''-terpyridine

Tetrafluoroborate salts of cationic ruthenium complexes [Ru(kappa(3)-tpy)(EPh(3))(2)Cl](+) (tpy=2,2':6',2'-terpyridine; E=P, 1 or As, 2) containing both the group 15 donor ligands and tpy and their representative substitution products are reported. Weak interaction {C-H...X (X=Cl, F and pi) and pi-pi interaction} studies revealed the presence of a double helical motif in complex 1, while the complex 2 assumes a single helical motif. Intercalative mode of interaction of the complexes 1 and 2 with calf thymus DNA (ctDNA) has been supported by absorption titration studies.     

3'-Terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for VPgpUpU-primed positive-strand initiation

The 5' cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5' cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5'-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3' ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the cre(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3'AAUUUUGUC5' sequence at the 3' ends of negative-strand templates. Our results suggest that this sequence is the primary cis-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.     

Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis.     

Asymmetric auxin response precedes asymmetric growth and differentiation of asymmetric leaf1 and asymmetric leaf2 Arabidopsis leaves

We have analyzed the development of leaf shape and vascular pattern in leaves mutant for ASYMMETRIC LEAVES1 (AS1) or AS2 and compared the timing of developmental landmarks to cellular response to auxin, as measured by expression of the DR5:beta-glucuronidase (GUS) transgene and to cell division, as measured by expression of the cycB1:GUS transgene. We found that the earliest visible defect in both as1 and as2 first leaves is the asymmetric placement of auxin response at the distal leaf tip. This precedes visible changes in leaf morphology, asymmetric placement of the distal margin gap, formation of margin gaps along the leaf border, asymmetric distribution of marginal auxin, and asymmetry in cell division patterns. Moreover, treatment of developing leaves with either exogenous auxin or an auxin transport inhibitor eliminates asymmetric auxin response and subsequent asymmetric leaf development. We propose that the initial asymmetric placement of auxin at the leaf tip gives rise to later asymmetries in the internal auxin sources, which subsequently result in asymmetrical cell differentiation and division patterns.     

Crystal structure of a carbohydrate induced homodimer of phospholipase A2 from Bungarus caeruleus at 2.1A resolution

This is the first crystal structure of a carbohydrate induced dimer of phospholipase A(2) (PLA(2)). This is an endogenous complex formed between two PLA(2) molecules and two mannoses. It was isolated from Krait venom (Bungarus caeruleus) and crystallized as such. The complete amino acid sequence of PLA(2) was determined using cDNA method. Three-dimensional structure of the complex has been solved with molecular replacement method and refined to a final R-factor of 0.192 for all the data in the resolution range 20.0-2.1A. The presence of mannose molecules in the protein crystals was confirmed using dinitrosalicylic acid test and the molecular weight of the dimer was verified with MALDI-TOF. As indicated by dynamic light scattering and analytical ultracentrifugation the dimer was also stable in solution. The good quality non-protein electron density at the interface of two PLA(2) molecules enabled us to model two mannoses. The mannoses are involved extensively in interactions with protein atoms of both PLA(2) molecules. Some of the critical amino acid residues such as Asp 49 and Tyr 31, which are part of the substrate-binding site, are found facing the interface and interacting with mannoses. The structure of the complex clearly shows that the dimerization is caused by mannoses and it results in the loss of enzymatic activity.     

Filaments of the Ure2p prion protein have a cross-beta core structure

Formation of filaments by the Ure2 protein constitutes the molecular mechanism of the [URE3] prion in yeast. According to the "amyloid backbone" model, the N-terminal asparagine-rich domains of Ure2p polymerize to form an amyloid core fibril that is surrounded by C-terminal domains in their native conformation. Protease resistance and Congo Red binding as well as beta-sheet content detected by spectroscopy-all markers for amyloid-have supported this model, as has the close resemblance between 40 A N-domain fibrils and the fibrillar core of intact Ure2p filaments visualized by cryo-electron microscopy and scanning transmission electron microscopy. Here, we present electron diffraction and X-ray diffraction data from filaments of Ure2p, of N-domains alone, of fragments thereof, and of an N-domain-containing fusion protein that demonstrate in each case the 4.7A reflection that is typical for cross-beta structure and highly indicative of amyloid. This reflection was observed for specimens prepared by air-drying with and without sucrose embedding. To confirm that the corresponding structure is not an artifact of air-drying, the reflection was also demonstrated for specimens preserved in vitreous ice. Local area electron diffraction and X-ray diffraction from partially aligned specimens showed that the 4.7A reflection is meridional and therefore the underlying structure is cross-beta.     

The efficiency of protein compartmentalization into the secretory pathway

Numerous proteins targeted for the secretory pathway are increasingly implicated in functional or pathological roles at alternative cellular destinations. The parameters that allow secretory or membrane proteins to reside in intracellular locales outside the secretory pathway remain largely unexplored. In this study, we have used an extremely sensitive and quantitative assay to measure the in vivo efficiency of signal sequence-mediated protein segregation into the secretory pathway. Our findings reveal that segregation efficiency varies tremendously among signals, ranging from >95 to <60%. The nonsegregated fraction is generated by a combination of mechanisms that includes inefficient signal-mediated translocation into the endoplasmic reticulum and leaky ribosomal scanning. The segregation efficiency of some, but not other signal sequences, could be influenced in cis by residues in the mature domain or in trans by yet unidentified cellular factors. These findings imply that protein compartmentalization can be modulated in a substrate-specific manner to generate biologically significant quantities of cytosolically available secretory and membrane proteins.     

Tumor necrosis factor alpha-mediated activation of c-Jun NH(2)-terminal kinase as a mechanism for fumonisin B(1) induced apoptosis in murine primary hepatocytes

Fumonisin B(1) is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species-specific and organ-specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B(1) perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B(1) caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B(1) induced hepatic apoptosis in mice. The role of tumor necrosis factor alpha (TNFalpha) in fumonisin B(1) mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B(1) induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B(1) caused accumulation of sphingoid bases and led to increase in TNFalpha expression. Phosphorylated and total c-Jun NH(2)-terminal kinase (JNK) activities were increased after 24 h fumonisin B(1) treatment. JNK inhibitor (SP600125) and anti-TNFalpha reduced the apoptosis induced by fumonisin B(1). The role of JNK signaling in fumonisin B(1) induced apoptosis is downstream of TNFalpha production, as fumonisin B(1)-mediated activation of JNK was reduced by the presence of anti-TNFalpha in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B(1) induced TNFalpha expression. Results of this study imply that generation of fumonisin B(1) induced TNFalpha results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes.     

Polypyrimidine tract binding protein blocks the 5' splice site-dependent assembly of U2AF and the prespliceosomal E complex

Polypyrimidine tract binding protein (PTB) represses some alternatively spliced exons by direct occlusion of splice sites. In repressing the splicing of the c-src N1 exon, we find that PTB acts by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5' splice site. Instead, PTB prevents formation of the prespliceosomal early (E) complex across the intervening intron by preventing the assembly of the splicing factor U2AF on the 3' splice site of exon 4. When the unregulated 5' splice site of the upstream exon 3 is present, U2AF binding is restored and splicing between exons 3 and 4 proceeds in spite of the N1 exon bound PTB. Thus, rather than directly blocking the N1 splice sites, PTB prevents the 5' splice site-dependent assembly of U2AF into the E complex. This mechanism likely occurs in many other alternative exons.     

Influence of salts and alcohols on the conformation of partially folded intermediate of stem bromelain at low pH

The effect of salts and alcohols was examined on the partially folded intermediate (PFI) state of stem bromelain reported at low pH (Haq, Rasheedi, and Khan (2002) European Journal of Biochemistry 269, 47-52) by a combination of optical methods like circular dichroism, intrinsic fluorescence and ANS binding. ESI mass spectrometry was also performed to see the effect, if any, on the overall tertiary structure of the protein. Increase in ionic strength by the addition of salts resulted in folded structures somewhat different from the native enzyme. Salt-induced intermediates are characterized by increase in helical content and a significantly reduced exposure of hydrophobic clusters relative to the state at pH 2.0. The emission wavelength maximum of intrinsic fluorescence was shifted towards that of native enzyme. ESI-MS data show decreased accessibility of ionizable/protonation sites suggestive of a folded structure. On the other hand, alcohol-induced intermediates though exhibiting increased helical content are apparently largely unfolded as observed by ESI. Thermal denaturation of a representative intermediate, each from the group of salts and alcohols examined, was also performed to check their relative stabilities. While the alcohol-induced state showed a cooperative thermal transition, the salt-induced state shows non-cooperative thermal denaturation.