E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO2 or 4-NO2 positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO2 group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs

Microarray analysis revealed different gene expression patterns in HepG2 cells treated with low and high concentrations of the extracts of Anacardium occidentale shoots

In this study, the effects of low and high concentrations of the Anacardium occidentale shoot extracts on gene expression in liver HepG2 cells were investigated. From MTT assays, the concentration of the shoot extracts that maintained 50% cell viability (IC50) was 1.7 mg/ml. Cell viability was kept above 90% at both 0.4 mg/ml and 0.6 mg/ml of the extracts. The three concentrations were subsequently used for the gene expression analysis using Affymetrix Human Genome 1.0 S.T arrays. The microarray data were validated using real-time qRT–PCR. A total of 246, 696 and 4503 genes were significantly regulated (P < 0.01) by at least 1.5-fold in response to 0.4, 0.6 and 1.7 mg/ml of the extracts, respectively. Mutually regulated genes in response to the three concentrations included CDKN3, LOC100289612, DHFR, VRK1, CDC6, AURKB and GABRE. Genes like CYP24A1, BRCA1, AURKA, CDC2, CDK2, CDK4 and INSR were significantly regulated at 0.6 mg/ml and 1.7 mg but not at 0.4 mg/ml. However, the expression of genes including LGR5, IGFBP3, RB1, IDE, LDLR, MTTP, APOB, MTIX, SOD2 and SOD3 were exclusively regulated at the IC50 concentration. In conclusion, low concentrations of the extracts were able to significantly regulate a sizable number of genes. The type of genes that were expressed was highly dependent on the concentration of the extracts used

Mechanism of cellular rejection in transplantation

The explosion of new discoveries in the field of immunology has provided new insights into mechanisms that promote an immune response directed against a transplanted organ. Central to the allograft response are T lymphocytes. This review summarizes the current literature on allorecognition, costimulation, memory T cells, T cell migration, and their role in both acute and chronic graft destruction. An in depth understanding of the cellular mechanisms that result in both acute and chronic allograft rejection will provide new strategies and targeted therapeutics capable of inducing long-lasting, allograft-specific tolerance

Functional analysis of a novel ENU-induced PHD finger 11 (Phf11) mouse mutant

Previously, human genetic studies have shown association between polymorphisms within the gene encoding plant homeodomain zinc finger protein 11 (PHF11) and asthma-related phenotypes. Initial functional studies have suggested that PHF11 may be involved in the immune response through regulation of T cell activities. In order to study further the gene’s functions, we have investigated the mouse Phf11 locus. We have established and characterised a mouse line harbouring a point mutation in the PHD domain of Phf11. Full-length mouse cDNA for Phf11 was obtained by applying rapid amplification of cDNA ends (RACE). All five exons encoding the PHD domain of Phf11 were directly sequenced in 3840 mouse DNA samples from the UK MRC Harwell ENU (N-ethyl-N-nitrosourea)-mutagenised DNA archive. Mice harbouring a valine to alanine substitution, predicted to have a significant functional impact on the PHD zinc finger domain, were re-derived. These Phf11 mutant mice were outcrossed to C3H mice and then backcrossed for ten generations in order to establish a congenic line harbouring the single point mutation in Phf11. Macroscopic examination, haematology and histological examination of lung structure revealed no significant differences between mutant and wild-type mice. After administration of lipopolysaccharide, the level of expression of Il2, NF-kB and Setdb2 were significantly increased in Phf11 mutant homozygous lungs compared to control littermates. Our results provide evidence that Phf11 can operate as a Th1 cell regulator in immune responses. Moreover, our data indicate that these mice may provide a useful model for future studies on Phf11. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00335-014-9535-x) contains supplementary material, which is available to authorised users

Discriminant Profile of Young Internet Dependents: The Role of Family Relationships

AbstractInternational studies have shown effects of family relations on Internet addiction in young people. This research aimed to outline a discriminant profile of young people classified as dependent and not dependent on the Internet regarding to socio-biodemographic variables to parenting practices, parent-child conflict and interparental conflict. The sample consisted of 200 students (152 girls and 48 boys), between 15 and 24 years of age, 85.5% reside in Rio Grande do Sul and 14.5% in other Brazilian states. Participants responded individually to the protocol available online. The results showed that interparental conflict, parent-child conflict and the educational practice of supervision of paternal behavior discriminate dependents on Internet. The educational practice of maternal emotional support was the only discriminating variable for non-dependents. These national findings corroborate the international context studies and reinforce the importance of including the family in promotion and prevention of mental health of young people