Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F.

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NFκB and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NFκB dependent since it was blocked by inhibitors of NFκB activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NFκB inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells.

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-γ treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells.

Infection of human endothelial cells by Japanese encephalitis virus: increased expression and release of soluble HLA-E.

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-β and TNF-α production.

Type IA topoisomerase inhibition by clamp closure.

Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration.

Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium.

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e.

Japanese encephalitis virus utilizes the canonical pathway to activate NF-kappaB but it utilizes the type I interferon pathway to induce major histocompatibility complex class I expression in mouse embryonic fibroblasts.

Flaviviruses have been shown to induce cell surface expression of major histocompatibility complex class I (MHC-I) through the activation of NF-kappaB. Using IKK1(-/-), IKK2(-/-), NEMO(-/-), and IKK1(-/-) IKK2(-/-) double mutant as well as p50(-/-) RelA(-/-) cRel(-/-) triple mutant mouse embryonic fibroblasts infected with Japanese encephalitis virus (JEV), we show that this flavivirus utilizes the canonical pathway to activate NF-kappaB in an IKK2- and NEMO-, but not IKK1-, dependent manner. NF-kappaB DNA binding activity induced upon virus infection was shown to be composed of RelA:p50 dimers in these fibroblasts.

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants.

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition.

A distinct physiological role of MutY in mutation prevention in mycobacteria.

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair.

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria.

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+ fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response.

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a model antigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.

Nonclassical MHC-I and Japanese encephalitis virus infection: induction of H-2Q4, H-2T23 and H-2T10.

Nonclassical MHC Class 1b antigens differ from classical MHC class 1a antigens in having a restricted polymorphism as well as varied surface expression in different cell types. They have been hypothesized to play a role in bridging adaptive and innate immune responses. We examined the effects of JEV infection on the expression of classical MHC class 1a and nonclassical MHC class 1b genes in five different cell lines.

Induction of classical and nonclassical MHC-I on mouse brain astrocytes by Japanese encephalitis virus.

Infection with Flaviviruses upregulates the cell surface expression of MHC-I, MHC-II, ICAM-1 (CD54), VCAM-1 (CD106) and TAP proteins. Although all these studies have been confirmed using West Nile virus and other Flaviviruses, there are few reports that have examined the effects of Japanese encephalitis virus (JEV) infection directly on nonclassical and classical MHC expression in astrocytes. We show in this report that JEV infection of mouse brain astrocytes results in induction of the nonclassical MHC Class Ib genes, H-2T23, H-2Q4 and H-2T10 in addition to MHC-I, Type I (alpha/beta) IFNs, TAP-1, TAP-2, Tapasin, LMP-2, LMP-7 and LMP-10 but not IFNgamma, CD80, CD86 and MHC-II genes.

Importance of uracil DNA glycosylase in Pseudomonas aeruginosa and Mycobacterium smegmatis, G+C-rich bacteria, in mutation prevention, tolerance to acidified nitrite, and endurance in mouse macrophages.

Uracil DNA glycosylase (Ung (or UDG)) initiates the excision repair of an unusual base, uracil, in DNA. Ung is a highly conserved protein found in all organisms. Paradoxically, loss of this evolutionarily conserved enzyme has not been seen to result in severe growth phenotypes in the cellular life forms.