Dynamic changes and molecular analysis of cell death in the spinal cord of SJL mice infected with the BeAn strain of Theiler’s murine encephalomyelitis virus

Theiler’s murine encephalomyelitis (TME) is caused by the TME virus (TMEV) and represents an important animal model for multiple sclerosis (MS). Oligodendroglial apoptosis and reduced apoptotic elimination of encephalitogenic leukocytes seem to participate in autoimmune demyelination in MS. The present study quantified apoptotic cells in BeAn–TMEV-induced spinal cord white matter lesions at 14, 42, 98, and 196 days post infection (dpi) using immunostaining. Apoptotic cells were identified by transmission electron microscopy and double-immunofluorescence. The mRNA expression of apoptosis-related genes was investigated using microarray analysis. Oligodendroglial apoptosis was already detected in the predemyelinating phase at 14 dpi. Apoptotic cell numbers peaked at 42 dpi and decreased until 196 dpi partly due to reduced T cell apoptosis. In addition to genes involved in the classical pathways of apoptosis induction, microarray analysis detected the expression of genes related to alternative mechanisms of cell death such as pyroptosis, necroptosis, and endoplasmic reticulum stress. Consequently, oligodendroglial apoptosis is involved in the initiation of the TME demyelination process, whereas the development of apoptosis resistance of T cells potentially favors the maintenance of inflammation and myelin loss.     

Enhancing the chemiluminescence intensity of a KMnO4 formaldehyde system for estimating the total phenolic content in honey samples using a novel nanodroplet mixing approach in a microfluidics platform

A novel mixing approach was utilized with a highly sensitive chemiluminescence (CL) method to determine the total phenolic content (TPC) in honey samples using an acidic potassium permanganate–formaldehyde system. The mixing approach was based on exploiting the mixing efficiency of nanodroplets generated in a microfluidic platform. Careful optimization of the instrument setup and various experimental conditions were employed to obtain excellent sensitivity. The mixing efficiency of the droplets was compared with the CL signal intensity obtained using the common serpentine chip design, with both approaches using at a total flow rate of 15 μl min^?1; the results showed that the nanodroplets provided 600% higher CL signal intensity at this low flow rate. Using the optimum conditions, calibration equations, limits of detection (LOD) and limits of quantification (LOQ) for gallic acid (GA), caffeic acid (CA), kaempferol (KAM), quercetin (QRC) and catechin (CAT) were obtained. The LOD ranged from 6.2 ppb for CA to 11.0 ppb for QRC. Finally, the method was applied for the determination of TPC in several local and commercial honey samples.     

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability.     

Determination of ibuprofen in pharmaceutical formulations using time-resolved terbium-sensitized luminescence.

A sensitive and specific luminescence method for the determination of ibuprofen (IB) in pharmaceutical formulations in aqueous solution is described. The method is based on the luminescence sensitization of terbium (Tb(3+)) by formation of ternary complex with IB in the presence of tri-n-octylphosphine oxide (TOPO) and Tween-20 as surfactant. The luminescence signal for Tb-IB-TOPO is monitored at lambda(ex) = 229 nm and lambda(em) = 545 nm. Optimum conditions for the formation of the complex in aqueous system, were 16 mmol/L TRIS buffer, pH 5.7, TOPO 200 micromol/L and 15 micromol/L of Tb(3+), which allows for the determination of 9.7 x 10(-7) - 9.7 x 10(-6) mol/L IB with a detection limit of 1.2 x 10(-7) mol/L. The relative standard deviations of the method were <1.4%, indicating excellent reproducibility. The proposed method was successfully applied for the assays of IB in pharmaceutical formulations with average recoveries of 100.3-102.5%.     

Carbon monoxide promotes hypoxic pulmonary vascular remodeling

CO is a biologically active gas that produces cellular effects by multiple mechanisms. Because cellular binding of CO by heme proteins is increased in hypoxia, we tested the hypothesis that CO interferes with hypoxic pulmonary vascular remodeling in vivo. Rats were exposed to inspired CO (50 parts/million) at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA (ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed, and changes in gene and protein expression of smooth muscle alpha-actin (sm-alpha-actin), beta-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of HH, vascular pressure at constant flow and vessel wall thickness increased and lumen diameter of small arteries decreased significantly. The presence of CO, however, further increased both pulmonary vascular resistance (PVR) and the number of small muscular vessels compared with HH alone. CO + HH also increased vascular PCNA and nuclear ssDNA expression compared with hypoxia, suggesting accelerated cell turnover. CO in hypoxia downregulated sm-alpha-actin and strongly upregulated beta-actin. CO also increased lung HO activity and HO-1 mRNA and protein expression in small pulmonary arteries during hypoxia. These data indicate an overall propensity of CO in HH to promote vascular remodeling and increase PVR in vivo.     

Purification and properties of cobalamin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae

In this study, we investigated methionine synthase from Candida albicans (CaMET 6p) and Saccharomyces cerevisiae (ScMET 6p). We describe the cloning of CaMet 6 and ScMet 6, and the expression of both the enzymes in S. cerevisiae. CaMET 6p is able to complement the disruption of met 6 in S. cerevisiae. Following the purification of ScMET 6p and CaMET 6p, kinetic assays were performed to determine substrate specificity. The Michaelis constants for ScMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 108, 84, 95, and 13 microM, respectively. The Michaelis constants for CaMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 113, 129, 120, and 14 microM, respectively. Neither enzyme showed activity with CH(3)-H(4)PteGlu(1) as a substrate. We conclude that ScMET 6p and CaMET 6p require a minimum of two glutamates on the methyltetrahydrofolate substrate, similar to the bacterial metE homologs. The cloning, purification, and characterization of these enzymes lay the groundwork for inhibitor-design studies on the cobalamin-independent fungal methionine synthases.     

Carbon monoxide, oxidative stress, and mitochondrial permeability pore transition

The cellular effects of carbon monoxide (CO) are produced primarily by CO binding to iron or other transition metals, which may also promote prooxidant activities of the more reactive gases, oxygen and nitric oxide. We tested the hypothesis that prooxidant effects of CO deregulate the calcium-dependent mitochondrial pore transition (MPT), which disrupts membrane potential and releases apoptogenic proteins. Rats were exposed to either CO (50 ppm) or hypobaric hypoxia (HH) for 1, 3, or 7 days, and liver mitochondria harvested to study protein expression and sensitivity to MPT by calcium and oxidants. Both exposures induced hypoxia-sensitive protein expression: hypoxia-inducible factor 1alpha (HIF-1alpha), heme oxygenase-1 (HO-1), and manganese SOD (SOD2), but SOD2 induction was greater by CO than by HH, especially at 7 days. Relative to HH, CO also caused significant early mitochondrial oxidative and nitrosative stress shown by decreases in GSH/GSSG and increases in protein 3-nitrotyrosine (3-NT) and protein mixed disulfide formation. This altered MPT sensitivity to calcium through an effect on the "S-site," causing loss of pore protection by adenine nucleotides. By 7 days, despite continued CO, nitrosative stress decreased and adenine nucleotide protection was restored to preexposure levels. This is the first evidence of functional mitochondrial pore stress caused by CO independently of its hypoxic effect, as well as a compensatory response exemplifying a mitochondrial phenotype shift. The implications are that cellular CO can activate or deactivate mitochondria for initiation of apoptosis in vivo.