Role of Tissue Factor in Mycobacterium tuberculosis-Induced Inflammation and Disease Pathogenesis

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

Functions of cytochrome c in regulation of electron transfer and protein folding.

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.     

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant plants.     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: effect of botulinum toxin A

The effects of mechanoreceptor stimulation and subsequent ATP release in cyclophosphamide evoked chronic bladder inflammation was examined to demonstrate: (1) whether inflammation modulates ATP release from bladder urothelium and (2) whether intravesical botulinum toxin A administration inhibits urothelial ATP release, a measure of sensory nerve activation. ATP release was measured from rat bladders in a Ussing chamber, an apparatus that allows one to separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Cystometry was utilized to correlate changes in ATP release with alterations in the frequency of voiding and non-voiding bladder contractions, in vivo measures of bladder afferent activity. The resting urothelial release of ATP was not significantly affected by either cyclophosphamide or botulinum toxin A treatment. However, evoked ATP release following hypoosmotic stimulation was significantly increased (i.e. 94%) in chronic cyclophosphamide treated bladder urothelium compared to control bladders. In addition, botulinum toxin A treatment significantly reduced hypoosmotic shock induced ATP release in cyclophosphamide treated animals by 69%. Cystometry revealed that cyclophosphamide and botulinum toxin A treatments altered non-voiding (i.e. cyclophosphamide increased, botulinum toxin A decreased) but not voiding contraction frequency suggesting that alterations in urothelial ATP release selectively diminished underlying bladder C-fiber nerve activity. Finally, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side implying that its effects were confined to the urothelial side of the bladder preparation.     

The expression of CCL2 by T lymphocytes of mammary tumor bearers: role of tumor-derived factors

Tumor-associated chemokines, including CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2), are thought to play many roles in cancer progression. Here we demonstrate the novel finding that during growth of the D1-7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is a dramatic up-regulation of CCL2 in splenic T cells at both the mRNA and protein levels upon stimulation. Of particular relevance is the finding that tumor-infiltrating T cells also produce high levels of CCL2. While a variety of tumor cell lines have been found to produce CCL2, we found no detectable levels of CCL2 protein in supernatants of the cultured mammary tumor cells. Investigation of the mechanisms involved in CCL2 induction showed that treatment of splenic T cells with the tumor-derived factors GM-CSF and phosphatidyl serine (PS) resulted in increased CCL2 production. This increased production may be involved in the downregulation of IFN-gamma by the T cells of tumor-bearing mice previously reported in this model, as treatment of splenic T lymphocytes with CCL2 resulted in a decreased secretion of IFN-gamma by those cells.     

Effects of dietary combination of chromium and biotin on growth performance, carcass characteristics, and oxidative stress markers in heat-distressed Japanese quail

Environmental stress causes adverse effects in performance and antioxidant status of poultry. Dietary chromium supplementation promotes the growth rate and feed efficiency of growing poultry and these beneficial effects of chromium appear to be greater under stress. Biotin, a member of the vitamin B complex, is involved in the metabolism of carbohydrates, fats, and proteins. In a previous experiment, we examined the effects of chromium picolinate (CrPic) as a chromium source in birds subjected to high environmental temperature and the data showed that supplementation with CrPic ameliorated the deletorious effect of stress. The study was conducted to determine the effects of a supplementation of combination of CrPic and biotin (Diachrome^TM) on performance, carcass characteristics, levels of oxidative stress markers, serum cholesterol, and glucose concentrations in Japanese quail (Coturnix coturnix japonica) exposed to high ambient temperature of 34°C. Two hundred forty Japanese quail (10 d old) were randomly assigned to 8 treatment groups consisting of 10 replicates of 3 birds. The birds were kept in a temperature-controlled room at 22°C (thermo-neutral [TN] groups) or 34°C (for 8h/d; 09.00 am to 05.00 pm; heat-stress [HS] groups). Birds were fed either a basal (control) diet (TN and HS) or the basal diet supplemented with either 1, 2 or 4 mg of Diachrome/kg of diet. Heat exposure decreased performance when the basal diet was fed (p=0.001). Diachrome supplementation increased feed intake (p=0.001), body weight (p=0.05), feed efficiency (p=0.01), and carcass traits (p≤0.05) variables linearly in birds reared under HS conditions. Serum vitamin C (p=0.05) and vitamin E (p=0.03) concentrations increased, whereas malondialdehyde (MDA) levels in serum and the liver (p=0.01), thigh muscle (p=0.05), and serum cholesterol and glucose concentrations (p=0.05) decreased in supplemented birds reared at a high temperature. It should be noted that when birds were kept at the thermo-neutral temperature, Diachrome supplementation did not affect (p>0.05) the variables measured, with the exception of a reduction in serum cholesterol and glucose. Results of the present study suggest that Diachrome can be considered a protective dietary supplement by reducing the negative effects of high environment temperature on performance and oxidative stress in quail.     

Fluorescence studies suggest a role for alpha-synuclein in the phosphatidylinositol lipid signaling pathway

Alpha-synuclein plays a key role in the pathogenesis of many neurodegenerative diseases. To date, its cellular role has yet to be determined, although it has been proposed to be connected to calcium and G protein-mediated dopamine signaling. Alpha-synuclein is known to bind strongly to model membrane surfaces where it may interact with other membrane-associated proteins. Here, we find that the membrane association of alpha-synuclein is enhanced by the presence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and Ca(2+). We also find that alpha-synuclein interacts with high affinity with the G protein-regulated enzyme phospholipase Cbeta(2) (PLCbeta(2)), which catalyzes the hydrolysis of PI(4,5)P(2). Binding of alpha-synuclein to PLCbeta(2) reduces its catalytic activity by 50%, but causes its level of activation by Gbetagamma subunits to increase from 4- to 24-fold. This effect is greatly reduced for A53T alpha-synuclein, which is a mutant associated with familial Parkinson's disease. PI(4,5)P(2) hydrolysis by PLCbeta(2) results in an increase in the intracellular Ca(2+) concentration, and we find that in cultured cells the presence of alpha-synuclein results in a 6-fold enhancement in the release of Ca(2+) from intracellular stores in response to agents that release Gbetagamma subunits relative to controls. Alpha-synuclein also enhances the increase in the level of inositol phosphates seen upon G protein stimulation, suggesting that it also may interact with PLCbeta(2) in cells. Given that Ca(2+) and dopamine regulation are mediated through PLCbeta and G protein signals, our results suggest that alpha-synuclein may play a role in inositol phospholipid signaling.