Staphylococcal alpha-toxin provokes neutrophil-dependent cardiac dysfunction: role of ICAM-1 and cys-leukotrienes

The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.     

Calcineurin and NFAT4 induce chondrogenesis

Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Here we show that elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. This suggested that calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Furthermore, activated NFAT4 induced BMP2 gene expression. These results have important implications for the effects of NFATs during development and adaptive responses.     

Phospholipid transfer protein deficiency protects circulating lipoproteins from oxidation due to the enhanced accumulation of vitamin E

Vitamin E is a lipophilic anti-oxidant that can prevent the oxidative damage of atherogenic lipoproteins. However, human trials with vitamin E have been disappointing, perhaps related to ineffective levels of vitamin E in atherogenic apoB-containing lipoproteins. Phospholipid transfer protein (PLTP) promotes vitamin E removal from atherogenic lipoproteins in vitro, and PLTP deficiency has recently been recognized as an anti-atherogenic state. To determine whether PLTP regulates lipoprotein vitamin E content in vivo, we measured alpha-tocopherol content and oxidation parameters of lipoproteins from PLTP-deficient mice in wild type, apoE-deficient, low density lipoprotein (LDL) receptor-deficient, or apoB/cholesteryl ester transfer protein transgenic backgrounds. In all four backgrounds, the vitamin E content of very low density lipoprotein (VLDL) and/or LDL was significantly increased in PLTP-deficient mice, compared with controls with normal plasma PLTP activity. Moreover, PLTP deficiency produced a dramatic delay in generation of conjugated dienes in oxidized apoB-containing lipoproteins as well as markedly lower titers of plasma IgG autoantibodies to oxidized LDL. The addition of purified PLTP to deficient plasma lowered the vitamin E content of VLDL plus LDL and normalized the generation of conjugated dienes. The data show that PLTP regulates the bioavailability of vitamin E in atherogenic lipoproteins and suggest a novel strategy for achieving more effective concentrations of anti-oxidants in lipoproteins, independent of dietary supplementation.     

Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120

The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(Delta 28)) or the hydrophobic and the linker region (MnSOD(Delta 60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 +/- 90) unit mg-1 protein and a dissociation constant for the inhibitor azide of (0.84 +/- 0.05) mm. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(Delta 28)) or (MnSOD(Delta 60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 x 10(6) m-1 s-1 (pH 10) and 6 x 10(7) m-1 s-1 (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria.     

A specific binding site for a fragment of the B-loop of epidermal growth factor and related peptides

Fragments of the B-loop of the epidermal growth factor family of peptides are reported to have mitogenic and angiogenic properties but appear to fail to compete with radioiodinated EGF in receptor binding. In this study, 11 analogs of a fragment of the B-loop of EGF-related peptides from several species were synthesized to study binding to A431 human epidermoid carcinoma using both 125I-EGF and [3'4'-3H-Tyr(22,29), Abu(20,31)]EGF(20-31)-NH(2). Specific binding sites were found for the human fragment and 8 analogs at a density five times higher than that of the EGF receptors. Analogs did not compete with 125I-EGF for binding to the EGF receptor. The novel binding site may mediate the biological effects of the fragments. The primary rather than secondary structure of the fragments appears to determine affinity.     

Sensitivity of whole body protein synthesis to amino acid administration during short-term bed rest

We tested the hypothesis that a reduced stimulation of whole-body protein synthesis by amino acid administration represents a major mechanism for the bed rest-induced loss of lean body mass. Healthy young subjects and matched controls were studied on the last day of a 14-day bed rest or ambulatory period, as part of the overall protocol "Short-term Bed Rest - Integrated Physiology" set up by the German Aerospace Centre (DLR) in co-operation with the European Space Agency. A balanced mixture of essential and non-essential amino acids was intravenously infused in the postabsorptive state for 3 hours at the rate of 0.1 g/kg/hour. The oxidative and non-oxidative (i.e., to protein synthesis) disposal of the infused leucine was determined by stable isotope and mass spectrometry techniques. The clearance of total infused amino acids tended to be greater (P=0.07) in the ambulatory group than in the bed rest group. When leucine clearance was partitioned between its oxidative and non-oxidative (i.e., to protein synthesis) components, the results indicated that the oxidative disposal was not statistically different in the bed rest and in the ambulatory groups. In contrast, the non-oxidative leucine disposal (i.e., to protein synthesis) was about 20% greater (P<0.01) in the ambulatory group than in the bed rest group. In conclusion, these preliminary data suggest that 14-day bed rest impairs the ability to utilise exogenous amino acids for protein synthesis.     

Effects of nicotine exposure on T cell development in fetal thymus organ culture: arrest of T cell maturation

There is evidence for both physiological functions of the natural neurotransmitter, acetylcholine, and pharmacological actions of the plant alkaloid, nicotine, on the development and function of the immune system. The effects of continuous exposure to nicotine over a 12-day course of fetal thymus organ culture (FTOC) were studied, and thymocytes were analyzed by flow cytometry. In the presence of very low concentrations of nicotine many more immature T cells (defined by low or negative TCR expression) and fewer mature T cells (intermediate or high expression of TCR) were produced. In addition, the numbers of cells expressing CD69 and, to a lesser extent, CD95 (Fas) were increased. These effects took place when fetal thymus lobes from younger (13-14 days gestation) pups were used for FTOC. If FTOC were set up using tissue from older (15-16 days gestation pups), nicotine had little effect, suggesting that it may act only on immature T cell precursors. Consistent with an increase in immature cells, the expression of recombinase-activating genes was found to be elevated. Nicotine effects were partially blocked by the simultaneous addition of the nicotinic antagonist d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of both immature and mature murine thymocytes, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors on developing thymocytes and influence the course of normal thymic ontogeny.     

Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.)

Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 micro g ml(-1) nutrient solution. At 10 micro g ml(-1), the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 micro g ml(-1), as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin-conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 microg ml(-1), however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation-inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 micro g ml(-1) for naringenin, hesperetin, and quercetin, respectively.     

Extracellular matrix proteins influence phenotype and cytokine expression in human breast cancer cell lines

Tumor growth and invasion are not only the result of malignant transformation but are also dependent on environmental influences from surrounding stroma, extracellular matrix (ECM), local cytokines and systemic hormones. We have investigated the influence of ECM components on three human breast cancer cell lines of different malignant potential: MCF-7, T47D and MDA-MB-231 were cultured on collagen I, collagen IV, laminin, fibronectin or poly-D-lysine, and we analyzed the proliferation rate and cytokine expression pattern. Among the three cell lines investigated we observed a distinct response to each ECM component. We hypothesize that ECM may have a significant modulatory effect on malignant behavior in vivo which might depend on individual responses and on the differentiation state of tumor cells. This study also shows that the surface on which cells are cultured greatly influences cell kinetics and the cytokine expression pattern.