Mechanism of Adsorption and Eclipse of Bacteriophage φX174 III. Comparison of the Activation Parameters for the In Vitro and In Vivo Eclipse Reactions With Mutant and Wild-Type Virus

In a starvation buffer containing 10(-3) M divalent cations, phiX174 undergoes viral eclipse above 20 C when attached to intact host cells. An in vitro structural transition that is similar to that observed in this in vivo eclipse reaction occurs over the same temperature range in 0.1 M CaCl(2) (pH 7.2). Since both reactions result in a loss of infectivity, their kinetics have been compared in this report. Both exhibit a biphasic first-order loss in PFU that is a result of two competing first-order processes. However, a single type of heterogeneity in the population of virions is not the basis for both competing slower reactions. The Arrhenius plots of the faster components show that the in vitro eclipse reaction has the same activation energy of 35 kcal/mol (ca. 1.47 x 10(5) J/mol) as the in vivo reaction but a 10-fold lower Arrhenius preexponential factor. This is further evidence that certain features of the in vivo mechanism are retained in the in vitro reaction. In the case of the slower components, the in vitro reaction has an activation energy of 37 kcal/mol (1.55 x 10(5) J/mol), whereas that of the in vivo reaction is only 5 kcal/mol (2.1 x 10(4) J/mol). A similar analysis has been performed on a cold-sensitive eclipse mutant of phiX174. In vivo, the mutation is expressed by a two- to three-fold lower Arrhenius preexponential factor for both components of the eclipse reaction when compared to wt virus. The activation energies for both components are the same as wt virus. These results suggest that the mechanism of the eclipse reaction can be operationally divided into two aspects, each subject to mutational alteration.     

Integrated In Silico–In Vitro Identification and Characterization of the SH3-Mediated Interaction between Human PTTG and its Cognate Partners in Medulloblastoma

The human pituitary tumor-transforming gene is an oncogenic protein which serves as a central hub in the cellular signaling network of medulloblastoma. The protein contains two vicinal PxxP motifs at its C terminus that are potential binding sites of peptide-recognition SH3 domains. Here, a synthetic protocol that integrated in silico analysis and in vitro assay was described to identify the SH3-binding partners of pituitary tumor-transforming gene in the gene expression profile of medulloblastoma. In the procedure, a variety of structurally diverse, non-redundant SH3 domains with high gene expression in medulloblastoma were compiled, and their three-dimensional structures were either manually retrieved from the protein data bank database or computationally modeled through bioinformatics technique. The binding capability of these domains towards the two PxxP-containing peptides m1p: ¹⁶¹LGPPSPVK¹⁶⁸ and m2p: ¹⁶⁸KMPSPPWE¹⁷⁵ of pituitary tumor-transforming gene were ranked by structure-based scoring and fluorescence-based assay. Consequently, a number of SH3 domains, including MAP3K and PI3K, were found to have moderate or high affinity for m1p and/or m2p. Interestingly, the two overlapping peptides exhibits a distinct binding profile to these identified domain partners, suggesting that the binding selectivity of m1p and m2p is optimized across the medulloblastoma expression spectrum by competing for domain candidates. In addition, two redesigned versions of m1p peptide ware obtained via a structure-based rational mutation approach, which exhibited an increased affinity for the domain as compared to native peptide.     

Characterization of MCU-Binding Proteins MCUR1 and CCDC90B — Representatives of a Protein Family Conserved in Prokaryotes and Eukaryotic Organelles

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Parallel Selections In Vitro Reveal a Preference for 2′–5′ RNA Ligation upon Deoxyribozyme-Mediated Opening of a 2′,3′-Cyclic Phosphate

We previously used in vitro selection to identify Mg²⁺-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5′-hydroxyl group with a 2′,3′-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2′–5′ phosphodiester bonds rather than native 3′–5′ linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2′–5′ linkages. This includes deoxyribozymes with an arrangement that favors 3′–5′ linkages for a different chemical reaction, that of a 2′,3′-diol plus 5′-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2′–5′ RNA linkages upon opening of a 2′,3′-cyclic phosphate with a 5′-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2′,3′-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.[Reviewing Editor: Niles Lehman]     

Design,Synthesis, and In Vitro and In Vivo Biological Studies of a 3′-Deoxythymidine Conjugate that Potentially Kills Cancer Cells Selectively

Thymidine kinases (TKs) have been considered one of the potential targets for anticancer therapeutic because of their elevated expressions in cancer cells. However, nucleobase analogs targeting TKs have shown poor selective cytotoxicity in cancer cells despite effective antiviral activity. 3′-Deoxythymidine phenylquinoxaline conjugate (dT-QX) was designed as a novel nucleobase analog to target TKs in cancer cells and block cell replication via conjugated DNA intercalating quinoxaline moiety. In vitro cell screening showed that dT-QX selectively kills a variety of cancer cells including liver carcinoma, breast adenocarcinoma and brain glioma cells; whereas it had a low cytotoxicity in normal cells such as normal human liver cells. The anticancer activity of dT-QX was attributed to its selective inhibition of DNA synthesis resulting in extensive mitochondrial superoxide stress in cancer cells. We demonstrate that covalent linkage with 3′-deoxythymidine uniquely directed cytotoxic phenylquinoxaline moiety more toward cancer cells than normal cells. Preliminary mouse study with subcutaneous liver tumor model showed that dT-QX effectively inhibited the growth of tumors. dT-QX is the first molecule of its kind with highly amendable constituents that exhibits this selective cytotoxicity in cancer cells.     

In Vitro Characterization of Echinomycin Biosynthesis: Formation and Hydroxylation of L-Tryptophanyl-S-Enzyme and Oxidation of (2S,3S) β-Hydroxytryptophan

Quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) feature in quinomycin family and confer anticancer activity. In light of the significant potency against cancer, the biosynthetic gene clusters have been reported from many different Streptomyces strains, and the biosynthetic pathway were proposed mainly based on the in vivo feeding experiment with isotope labeled putative intermediates. Herein we report another gene cluster from Streptomyces griseovariabilis subsp. bandungensis subsp. nov responsible for the biosynthesis of echinomycin (a member of quinomycin family, also named quinomycin A) and presented in vitro evidence to corroborate the previous hypothesis on QXC biosynthesis, showing that only with the assistance of a MbtH-like protein Qui5, did the didomain NRPS protein (Qui18) perform the loading of a L-tryptophan onto its own PCP domain. Particularly, it was found that Qui5 and Qui18 subunits form a functional tetramer through size exclusion chromatography. The subsequent hydroxylation on β-carbon of the loaded L-tryptophan proved in vitro to be completed by cytochrome P450-dependent hydroxylase Qui15. Importantly, only the Qui18 loaded L-tryptophan can be hydroxylated by Qui15 and the enzyme was inactive on free L-tryptophan. Additionally, the chemically synthesized (2S,3S) β-hydroxytryptophan was detected to be converted by the tryptophan 2,3-dioxygenase Qui17 through LC-MS, which enriched our previous knowledge that tryptophan 2,3-dioxygenase nearly exclusively acted on L-tryptophan and 6-fluoro-tryptophan.     

In vivo visualization of RNA in plants cells using the λN₂₂ system and a GATEWAY-compatible vector series for candidate RNAs

The past decade has seen a tremendous increase in RNA research, which has demonstrated that RNAs are involved in many more processes than were previously thought. The dynamics of RNA synthesis towards their regulated activity requires the interplay of RNAs with numerous RNA binding proteins (RBPs). The localization of RNA, a mechanism for controlling translation in a spatial and temporal fashion, requires processing and assembly of RNA into transport granules in the nucleus, transport towards cytoplasmic destinations and regulation of its activity. Compared with animal model systems little is known about RNA dynamics and motility in plants. Commonly used methods to study RNA transport and localization are time‐consuming, and require expensive equipment and a high level of experimental skill. Here, we introduce the λN₂₂ RNA stem‐loop binding system for the in vivo visualization of RNA in plant cells. The λN₂₂ system consists of two components: the λN₂₂ RNA binding peptide and the corresponding box‐B stem loops. We generated fusions of λN₂₂ to different fluorophores and a GATEWAY vector series for the simple fusion of any target RNA 5′ or 3′ to box‐B stem loops. We show that the λN₂₂ system can be used to detect RNAs in transient expression assays, and that it offers advantages compared with the previously described MS2 system. Furthermore, the λN₂₂ system can be used in combination with the MS2 system to visualize different RNAs simultaneously in the same cell. The toolbox of vectors generated for both systems is easy to use and promises significant progress in our understanding of RNA transport and localization in plant cells.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

Effect of Pepsin–Trypsin In Vitro Gastro-Intestinal Digestion on the Antioxidant Capacities of Ultra-Filtrated Rice Bran Protein Hydrolysates (Molecular Weight > 10 kDa; 3–10 kDa,and < 3 kDa)

International Journal of Peptide Research and Therapeutics - The antioxidant capacities of rice bran protein hydrolysates (RBPH) obtained by in vitro gastro-intestinal (GI) digestion by...     

DNMT3B polymorphisms and cancer risk: a meta analysis of 24 case–control studies

The association between polymorphism of DNA methyltransferases 3B and cancer risk has been widely studied recently, and no consensus conclusion is available up to now. We perform a comprehensive search using the databases of Medline, ISI Web of Knowledge and Embase. The odds ratio (OR) and its 95% confidence interval (95% CI) are used to investigate the strength of the association. A total of 24 case–control studies with 15,647 individuals are included in this meta-analysis. For −149C > T (17 studies, 5229 cases and 6910 controls), no evidence indicate that individuals carrying the variant genotypes (CC + CT), relative to those carrying the wild homozygote TT genotype, have an increased risk of cancer (OR = 1.03; 95% CI = 0.84–1.26; P = 0.76). Similarly, no cancer risk is found in the subgroup analyses. For −579G > T (11 studies, 3513 cases and 3714 controls), significantly decreased risks of cancer are observed, and the ORs (95% CI) are 0.70 (0.56–0.87) for GT versus TT, 0.70 (0.57–0.85) for GG + GT versus TT and 0.76 (0.63–0.93) for G-allele versus T-allele, respectively. Subgroup analyses stratified by ethnicity and types of cancer are also performed, and results indicated that −579G > C polymorphism is associated with risk of cancer in Asians [0.68 (0.53–0.87) for GT vs. TT] but not in Europeans [0.82 (0.63–1.07) for GT vs. TT]. We also observe that the −579G is associated with decreased risk of colorectal cancer [0.49(0.38–0.62) for GT vs. TT]. More studies with larger sample size were needed to provide more precise evidence.