Statistical modeling of cell-to-cell variability in viral infection during passaging in suspension cell culture: Application in Monte-Carlo simulation

Packaging during the passaging of viruses in cell cultures yields various phenotypes and is regulated by viral protein expression in infected cells. Although such a packaging mechanism has a profound effect in controlling the virus yield, little is known about the underlying statistical models followed by virus packaging and protein expression among cells infected with the virus. A predictive framework combining identification of the probability density function (PDF) based on log-likelihood and using the PDF for Monte-Carlo simulations is developed. The Birnbaum–Saunders distribution was found to be consistent with all three-virus packaging levels, including nucleocapsids/occlusion-derived virus (ODV), ODVs/polyhedra, and polyhedra/cell for both wild-type and genetically modified AcMNPV. Next, it was demonstrated that PDF fitting could be used to compare two viruses having distinctly different genetic configurations. Finally, the identified PDF can be incorporated in RNA synthesis parameters for baculovirus infection to predict the cell-to-cell variability in protein expression using Monte-Carlo simulations. The proposed tool can be used for the estimation of uncertainty in the kinetic parameter and prediction of cell-to-cell variability for other biological systems.     

Spectroscopic characterization of the NO adduct of hydroxylamine oxidoreductase

Hydroxylamine oxidoreductase (HAO) from the autotrophic nitrifying bacterium Nitrosomonas europaea catalyzes the oxidation of NH2OH to NO2-. The enzyme contains eight hemes per subunit which participate in catalysis and electron transport. NO is found to bind to the enzyme and inhibit electron flow to the acceptor protein, cytochrome c554. NO is found to oxidize either partially or fully reduced HAO, but NO will not reduce ferric HAO. Since NO can be reduced but not oxidized to product by HAO, NO is not considered to be a long-lived intermediate in the catalytic mechanism. Substrate oxidation occurs in the presence of bound NO or cyanide, suggesting a second interaction site for substrate with HAO and providing a means for recovery of the NO-inhibited form of the enzyme. Upon addition of NO to oxidized HAO, the integer-spin EPR signal from the active site vanishes, an IR band from NO appears at 1920 cm(-1), and a diamagnetic quadrupole iron doublet appears in M?ssbauer spectroscopy with delta = 0.06 mm/s and DeltaEq = 2.1 mm/s. The NO stretching frequency and M?ssbauer parameters are characteristic of an [FeNO]6 heme complex. New M?ssbauer data on ferric myoglobin-NO are also presented for comparison. The results indicate that NO binds to heme P460 and that the loss of the integer-spin EPR signal is due to the conversion of heme P460 to a diamagnetic S = 0 state and concomitant loss of magnetic interaction with neighboring heme 6. In previous studies where the heme P460-heme 6 interaction was affected by substrate or cyanide binding, a signal attributable to heme 6 was not observable. In contrast, in this work, the NO-induced loss of the signal is accompanied by the appearance of a previously unobserved large g(max) (or HALS) low-spin EPR signal from heme 6.     

Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis. Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases. It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded. As part of a study of B. pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78-261). Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE(78-261) is monomeric in aqueous solution. CD spectroscopy indicates that the protein is predominantly alpha-helical, with predicted secondary structure composition of 59% alpha-helix and 7% beta-strand. NMR spectroscopy confirms that BopE(78-261) adopts a single, stable conformation. In differential scanning calorimetry experiments, thermal denaturation of BopE(78-261) (T(m) 52 degrees C) is reversible. Also, the secondary structure composition of BopE(78-261) changes little over a range of pH values from 3.5 to 10.5. BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments. These properties are likely to be advantageous for a secreted bacterial effector protein.     

Fibroblast growth factor-10 upregulates Na,K-ATPase via the MAPK pathway

We studied the effects of fibroblast growth factor (FGF-10) on alveolar epithelial cell (AEC) Na,K-ATPase regulation. Within 30 min FGF-10 increased Na,K-ATPase activity and alpha1 protein abundance by 2.5-fold at the AEC plasma membrane. Pretreatment of AEC with the mitogen-activated protein kinase (MAPK) inhibitor U0126, a Grb2-SOS inhibitor (SH3-b-p peptide), or a Ras inhibitor (farnesyl transferase inhibitor (FTI 277)), as well as N17-AEC that express a Ras dominant negative protein each prevented FGF-10-mediated Na,K-ATPase recruitment to the AEC plasma membrane. Accordingly, we provide first evidence that FGF-10 upregulates (short-term) the Na,K-ATPase activity in AEC via the Grb2-SOS/Ras/MAPK pathway.     

Nitric oxide protects against chloroquine resistant Plasmodium yoelii nigeriensis parasites in vitro

Malaria is a life-threatening disease of global concern. The role of nitric oxide in the clearance of malarial parasites is still under debate. Several reports suggest a possible role for nitric oxide in the protection during initial stages of malarial infection. In the present study, we demonstrate that the nitric oxide in combination with low concentrations of chloroquine controls the parasitaemia in vitro. Activated peritoneal macrophages co-cultured with lipopolysaccharide+interferon-gamma or extracts from Tenospora cordifolia as an immunomodulator promoted nitric oxide production by macrophages. The high concentration of nitric oxide in combination with sub-optimal chloroquine suppressed the parasitaemia in the chloroquine resistant malarial infection. Further, the nitric oxide synthase inhibitor, N(G)-mono-methyl-l-arginine, downregulated nitric oxide production by peritoneal macrophages and the resulting levels of parasitaemia were higher, similar to those of untreated controls. These findings support the proposition that nitric oxide has a crucial role in the control of parasitaemia at the initial periods of blood stage malarial infection.     

Studies on the immunogenic potential of plant-expressed cholera toxin B subunit

Nicotiana tabacum var. Samsun was transformed via Agrobacterium-mediated transformation with a gene encoding the cholera toxin B subunit (CTB) of Vibrio cholerae, modified to contain a sequence coding for an endoplasmic reticulum retention signal (SEKDEL), under the control of the cauliflower mosaic virus 35S promoter. Total protein from the transgenic leaf tissue was isolated and an aliquot containing 5 g recombinant CTB was injected intradermally into Balb/c (H2K^d) mice. CTB-specific serum IgG was detected in animals that had been administered plant-expressed or native purified CTB. A T-cell proliferation study using splenocytes and cytokine estimations in supernatants generated by in vitro stimulation of macrophages isolated from the immuno-primed animals was carried out. Inhibition of proliferation of T lymphocytes was observed in splenic T lymphocytes isolated from animals injected with either native or plant-expressed CTB. Macrophages isolated from mice immunised with native or plant-expressed CTB showed enhanced secretion of interleukin-10 but secretion of lipopolysaccharide-induced interleukin-12 and tumor necrosis factor alpha was inhibited. These studies suggest that plant-expressed protein behaved like native CTB with regards to effects on T-cell proliferation and cytokine levels, indicating the suitability of plant expression systems for the production of bacterial antigens, which could be used as edible vaccine. The transgene was found to be inherited in the progeny and was expressed to yield a pentameric form of CTB as evident by its interaction with G_M1 ganglioside.Abbreviations BAP 6-Benzylaminopurine - Con A Concanavilin A - CTB Cholera toxin B subunit - ctxB Gene encoding cholera toxin B subunit - ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - IL-10 Interleukin-10 - IL-12 Interleukin-12 - LPS Lipopolysaccharide - NAA Naphthaleneacetic acid - PBS Phosphate-buffered saline - TNF Tumour necrosis factor alphaCommunicated by H. Uchimiya     

Biphasic diurnal cycle of ascus development of Taphrina maculans Butler

Taphrina maculans Butler inciting the brown leaf spot disease of turmeric (Curcuma longa L.) produces cuboid ascogenous cells several layers in depth in the subcuticular interspaces of the epidermis. The ascogenous cells germinate to produce asci centrifugally maturing toward the periphery of the stroma, expelling octosporous microcolonies of asco-blastospores on the leaf surface inciting secondary infection in favorable environment. Occurrence of a rhythmic cycle of ascus development and ascospore discharge giving 2 peaks of ascospore discharge each day has been demonstrated in Taphrina maculans. The cycle is directly affected by atmospheric temperature, availability of free moisture on the leaf surface and sunlight. Free moisture on a leaf surface appropriately soaks the infection spots (ascogenous cells) and induces ascus elongation and ascospore discharge, when a suitable atmospheric temperature is reached. Sunlight may adversely affect the cycle by increasing the temperature and lowering humidity in the atmosphere.