Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer.

Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo.

A Novel AT-Rich DNA Recognition Mechanism for Bacterial Xenogeneic Silencer MvaT.

Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an "AT-pincer" motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA.

Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX.

We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands.

Oscillometrically measured blood pressure in Hong Kong Chinese children and associations with anthropometric parameters.

BACKGROUND: Oscillometric devices are increasingly used to measure blood pressure (BP). Reference data are limited and have not used devices validated against sphygmomanometric measurements on which current standards are based. BP standards for Chinese children have been based on sphygmomanometry and have not provided height-related or weight-related BP percentiles. METHODS: BP was measured in 14842 Hong Kong Chinese schoolchildren aged 6-18 years randomly selected from 36 schools in the 18 Hong Kong districts, using a validated oscillometric device (Datascope Accutorr Plus). Height, weight, heart rate and waist circumference were measured. Percentiles for systolic BP and diastolic BP by sex, age, height and weight were generated. Features associated with systolic BP and diastolic BP in 12680 children were analysed by univariate and multivariate analysis. RESULTS: Reference BP standards by sex, age, weight and height are presented. BP was associated (in descending order of strength) with weight > height > age > waist circumference > body mass index, and weakly with heart rate (which added considerable influence on multivariate analysis). BP increases similarly with age, height (which can normalize for variations in growth) and weight (which is associated most strongly with BP). BP was associated also with family history of high BP and (inversely) with sleep duration. CONCLUSIONS: The study provides oscillometrically measured BP standards for Chinese children, with age-related and sex-related height-specific and weight-specific percentiles. Implications of the findings are discussed. Screening by sex-specific BP-height percentile charts, and then if high, reference to the BP-sex-age-weight table, is suggested.

Structural changes accompanying human serum albumin's binding of fatty acids are concerted.

Long chain fatty acids (LCFAs), a major source of cellular energy, are solubilized and transported in the blood by binding to serum albumin. Changes in human serum albumin's (HSA's) UV absorption and characteristic reactivity with pyridoxal-5'-phosphate appear to reflect a concerted change in its structure upon binding five equivalents of myristate. Isothermal titrations with myristate and other LCFA anions are also consistent with the presence of five strong, interacting, binding sites. Although HSA is usually thought to have many independent LCFA anion binding sites, just five interacting sites appear to account for the changes in structure that accompany its binding of myristate.

Reactions of acrylamide with glutathione and serum albumin.

Rate constants of 0.0054 and 0.021 M(-1)s(-1) for the reactions of acrylamide with human serum albumin (HSA) and glutathione (GSH), respectively, were determined under physiological conditions by following the loss of their thiol groups in the presence of excess acrylamide. Based on these in vitro values, reactions with these thiols appear to account for most of acrylamide's elimination from the body. Combined with data from other studies, these results should be useful for assessing the health risk of dietary acrylamide.