Development of a Serum-free Suspension Process for the Production of a Conditionally Replicating Adenovirus using A549 Cells

Conditionally replicating adenoviruses (CRAVs) are a group of recombinant human adenoviruses genetically engineered to replicate in selected tissues, such as tumors. These viruses could potentially offer significant medicinal benefits, since the restrictive replication of these viral vectors leads to the lysis of target cells without harm to the surrounding tissues. The in vitro propagation and amplification of the CRAV vectors often requires special host cells with deregulated growth control pathways. In order to develop an efficient cell culture process for the scaleable production of a CRAV vector, A549 cells, a human lung carcinoma cell line normally cultured in adherent culture, were adapted to suspension culture. CRAV production was demonstrated with the suspension-adapted A549 cells and a baseline production process was developed in shake flasks. The ability to scale-up virus production was confirmed in stirred tank bioreactors. Molecular characterization of the suspension-adapted A549 cells indicates no significant changes in cellular mechanisms related to adenovirus infection.     

Human Rad51C deficiency destabilizes XRCC3, impairs recombination, and radiosensitizes S/G2-phase cells

The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) are expressed in mitotically growing cells and are thought to play mediating roles in homologous recombination, although their precise functions remain unclear. Among the five paralogs, Rad51C was found to be a central component present in two complexes, Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2. We have shown previously that the human Rad51C protein exhibits three biochemical activities, including DNA binding, ATPase, and DNA duplex separation. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA-cross-linking agent mitomycin C and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G(2)/M phases of the cell cycle but not in G(1) phase. Together, these results provide direct cellular evidence for the function of human Rad51C in homologous recombinational repair.     

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.      

Genetics of longevity. data from the studies on Sicilian centenarians

ABSTRACT: The demographic and social changes of the past decades have determined improvements in public health and longevity. So, the number of centenarians is increasing as a worldwide phenomenon. Scientists have focused their attention on centenarians as optimal model to address the biological mechanisms of "successful and unsuccessful ageing". They are equipped to reach the extreme limits of human life span and, most importantly, to show relatively good health, being able to perform their routine daily life and to escape fatal age-related diseases, such as cardiovascular diseases and cancer. Thus, particular attention has been centered on their genetic background and immune system. In this review, we report our data gathered for over 10 years in Sicilian centenarians. Based on results obtained, we suggest longevity as the result of an optimal performance of immune system and an over-expression of anti-inflammatory sequence variants of immune/inflammatory genes. However, as well known, genetic, epigenetic, stochastic and environmental factors seem to have a crucial role in ageing and longevity. Epigenetics is associated with ageing, as demonstrated in many studies. In particular, ageing is associated with a global loss of methylation state. Thus, the aim of future studies will be to analyze the weight of epigenetic changes in ageing and longevity.     

PTEN catalysis of phospholipid dephosphorylation reaction follows a two-step mechanism in which the conserved aspartate-92 does not function as the general acid--mechanistic analysis of a familial Cowden disease-associated PTEN mutation

PTEN exerts its tumour suppressor function by dephosphorylating the phospholipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP(3)). Herein, we demonstrate that the PTEN-catalysed PIP(3) dephosphorylation reaction involves two-steps: (i) formation of a phosphoenzyme intermediate (PE) in which Cys-124 in the active site is thiophosphorylated, and (ii) hydrolysis of PE. For protein tyrosine- and dual-specificity phosphatases, catalysis requires the participation of a conserved active site aspartate as the general acid in Step 1. Its mutation to alanine severely limits PE formation. However, mutation of the homologous Asp-92 in PTEN does not significantly limit PE formation, indicating that Asp-92 does not act as the general acid. G129E is a common germline PTEN mutations found in Cowden syndrome patients. Mechanistic analysis reveals that this mutation inactivates PTEN by both significantly slowing down Step 1 and abolishing the ability to catalyse Step 2. Taken together, our results highlight the mechanistic similarities and differences between PTEN and the conventional protein phosphatases and reveal how a disease-associated mutation inactivates PTEN.     

Polymorphisms of pro-inflammatory genes and prostate cancer risk: a pharmacogenomic approach

In this paper, we consider the role of the genetics of inflammation in the pathophysiology of prostate cancer (PCa). This paper is not an extensive review of the literature, rather it is an expert opinion based on data from authors’ laboratories on age-related diseases and inflammation. The aim is the detection of a risk profile that potentially allows both the early identification of individuals at risk for disease and the possible discovery of potential targets for medication. In fact, a major goal of clinical research is to improve early detection of age-related diseases, cancer included, by developing tools to move diagnosis backward in disease temporal course, i.e., before the clinical manifestation of the malady, where treatment might play a decisive role in preventing or significantly retarding the manifestation of the disease. The better understanding of the function and the regulation of inflammatory pathway in PCa may help to know the mechanisms of its formation and progression, as well as to identify new targets for the refinement of new treatment such as the pharmacogenomics approach.     

Immunization with an ApoB-100 Related Peptide Vaccine Attenuates Angiotensin-II Induced Hypertension and Renal Fibrosis in Mice

Recent studies suggest the potential involvement of CD8+ T cells in the pathogenesis of murine hypertension. We recently reported that immunization with apoB-100 related peptide, p210, modified CD8+ T cell function in angiotensin II (AngII)-infused apoE (-/-) mice. In this study, we hypothesized that p210 vaccine modulates blood pressure in AngII-infused apoE (-/-) mice. Male apoE (-/-) mice were immunized with p210 vaccine and compared to unimmunized controls. At 10 weeks of age, mice were subcutaneously implanted with an osmotic pump which released AngII for 4 weeks. At 13 weeks of age, p210 immunized mice showed significantly lower blood pressure response to AngII compared to controls. CD8+ T cells from p210 immunized mice displayed a different phenotype compared to CD8+ T cells from unimmunized controls. Serum creatinine and urine albumin to creatinine ratio were significantly decreased in p210 immunized mice suggesting that p210 vaccine had renal protective effect. At euthanasia, inflammatory genes IL-6, TNF-α, and MCP-1 in renal tissue were down-regulated by p210 vaccine. Renal fibrosis and pro-fibrotic gene expression were also significantly reduced in p210 immunized mice. To assess the role of CD8+ T cells in these beneficial effects of p210 vaccine, CD8+ T cells were depleted by CD8 depleting antibody in p210 immunized mice. p210 immunized mice with CD8+ T cell depletion developed higher blood pressure compared to mice receiving isotype control. Depletion of CD8+ T cells also increased renal fibrotic gene expression compared to controls. We conclude that immunization with p210 vaccine attenuated AngII-induced hypertension and renal fibrosis. CD8+ T cells modulated by p210 vaccine could play an important role in the anti-hypertensive, anti-fibrotic and renal-protective effect of p210 vaccine.     

Synthesis of Nh-acyl-α-aminoamides on rink resin: Inhibitors of the hematopoietic protein tyrosine phosphatase (HePTP)

Cinnamic acid derivatives were prepared on Rink resin using tert-butyl-4-carboxycinnamate in a four-component Ugi condensation. Inhibition of the hematopoietic protein tyrosine phosphatase (HePTP) by these small molecules is reported. An IC₅₀ value of 3.9 μM was determined for the most potent inhibitor discovered.