Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-κB pathway in lipopolysaccharide-stimulated macrophage

Quercetin is a flavonoid molecule ubiquitous in nature and functions as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. Dose- and time-dependent effect of quercetin has been investigated on proinflammatory cytokine expression and NO production, focusing on its effects on the MAP kinases and the NF-B signal transduction pathways in LPS-stimulated RAW 264.7 cells by using RT-PCR and immunoblotting. Quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase by LPS treatment. In addition, quercetin treatment inhibited NF-B activation through stabilization of the NF-B/IB complex and IB degradation and proinflammatory cytokines and NO/iNOS expression. Quercetin may exert its anti-inflammatory and immunomodulatory properties in the effect molecules such as proinflammatory cytokines and NO/iNOS by suppressing the activation of ERK and p38 MAP kinase, and NF-B/IB signal transduction pathways.     

Inhibition of NF-κ B activity and cFLIP expression contribute to viral-induced apoptosis

Virus-induced activation of nuclear factor-kappa B (NF-B) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-B is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-B activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-B regulation, where NF-B activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-B activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-B is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-B also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-B-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-B activation in reovirus-infected cells. Further, inhibition of NF-B activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-B and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis.     

Stimulation of proliferation and immunoglobulin production of human-human hybridoma by various types of caseins and their protease digests

We have studied the effect of such milk proteins as caseins, lactalbumin, and lactoglobulin, on proliferation and immunoglobulin production of human-human hybridoma HB4C5 cells. It was found that -, -, and -caseins stimulated both proliferation and IgM product ion of human-human hybridoma HB4C5 cells, while the activities of -lactalbumin and -lactoglobulin were negligible. To localize the active sites of these caseins, effect of protease treatments on the activities were examined. When caseins were digested with trypsin, casein digests stimulated proliferation of the hybridoma, but not their IgM production. When -casein was digested with chymosin and fractionated top--casein and glycomacropeptide, both fragments stimulated proliferation of the cells, but onlyp--casein fragment stimulated IgM production. These results indicate that -casein has at least two proliferation stimulating sites and an IgM production stimulating site in thep--casein region.     

Effect of gelation temperature and gel-dissolving solution on cell viability and recovery of two Pseudomonas putida strains co-immobilized within calcium alginate or κ-carrageenan gel beads

Summary Since a lethal effect of an increased temperature (42°C) on Pseudomonas putida strains PaW8 and PaW130 was demonstrated, strictly ionotropic gels such as calcium alginate or -carrageenan type X 0909 were used for cell co-immobilization, rather than a thermoionotropic -carrageenan gel. Among the variety of gel-dissolving solutions tested, only a 0.05M Na₂CO₃/0.02M citric acid solution was able to preserve around 100 % of the cell viability. A complete cell recovery was obtained from calcium alginate gel beads, while only 6 % of viable cells was recovered from the ionotropic -carrageenan gel.     

Quantitative recovery of fungal biomass grown on solid κ- carrageenan media

The use of a dissolvable solid matrix, -carrageenan, to quantify biomass grown on solid media was studied. A firm gel was obtained with 2% (w/v) -carrageenan and 20 mM K+ which could be easily dissolved in demineralized water. Direct quantification of Coniothyrium minitans biomass grown on this medium was feasible. No effects of the dissolution on the amount of biomass recovered were detected.     

Infrared analysis of eleven carrageenophytes from Baja California,Mexico

Infrared analyses of the carrageenan in ten species (representing four genera) of Gigartinaceae and one species of Hypneaceae in different reproductive phases from the northwestern coast of Baja California were studied. Cystocarpic samples of the Gigartinaceae presented varying degrees of a / hybrid. The degree of hybridization was determined based on the ratio between the peak absorbances at 805/845 cm^–1. A high correlation was observed between the 805/845 cm^–1 and 805/970 cm^–1 ratios. Tetrasporic samples of Gigartina leptorhynchos, Iridaea splendens, Rhodoglossum affine and R. roseum, presented a -carrageenan profile, whereas Gigartina tepida, G. exasperata, G. harveyana, G. canaliculata and G. spinosa presented a -carrageenan. The tetrasporic sample of Hypnea valentiae showed a -carrageenan with a very low degree of hybridization.     

Characterization ofκ-casein and keratin domains in fibrinogen

Summary Following the observation of a close sequence homology between the N-terminal moiety of the -chain of fibrinogen with large parts of-casein, the occurrence of a keratin domain in the middle section of the Achain is suggested.     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .     

The microbial production of 3aα-H-4α- (3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-indan-1-one-δ-lactone from cholesterol

Summary A mutant strain of Rhodococcus australis CSIR-236.457 which accumulates 3a-H-4-(3-propionic acid)-5-hydroxy-7a-methylhexahydro-indan-1-one--lactone from cholesterol, stigmasterol and -sitosterol was studied. The product is produced in a 60% molar yield in a dilute black strap molasses medium containing 6–12g/l cholesterol after a 72 hour fermentation period.     

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

Temperature effects on C- and N-mineralization from vegetable crop residues

Net N-mineralization and nitrification from soil organic matter and from vegetable crop residues (leaf-blades of cauliflower and stems of red cabbage) were measured at 4 temperatures during aerobic incubation in the laboratory. C-mineralization from leaf-blades of cauliflower was monitored at 3 different temperatures. N-mineralization from soil organic matter was best described by zero order kinetics N(t)=kt whereas N- and C-mineralization from the crop residues were described by single first order kinetics. Stems of red cabbage mineralized much more slowly than leaf-blades of cauliflower. S-shaped functions were fitted to the relationship between the rate constants of both C and N-mineralization and temperature. The rate parameter of the S-shaped function reflects the temperature dependence of the mineralization rate k. The parameter for N-mineralization of the stem material (=5.36) was significantly higher than for the leaf-blades (=3.38), indicating that there is a strong interaction between temperature and resistance to degradation in the soil. N-mineralization from soil organic matter was least sensitive to temperature (=2.63). Temperature dependence of nitrification was not significantly different from mineralization over the temperature range considered. Rate constants for C-mineralization of cauliflower leaf-blades were higher than for N-mineralization, but the temperature dependence of the rate constants was not significantly different for both processes.     

Role of Opiate Receptors and ATP-Dependent Potassium Channels of Mitochondria in the Formation of Myocardial Adaptive Resistance to the Arrhythmogenic Effect of Ischemia and Reperfusion

Preliminary stimulation of opiate receptors (ORs) by intravenous administration of agonist DALDA (0.5 mg/kg), ₁ agonist DPDPE (0.5 mg/kg), and agonist (-)-U-50.488 (1 mg/kg) increases rat myocardial resistance to arrhythmogenic effect of coronary occlusion (10 min) and reperfusion (10 min). Activation of ₂ ORs (DSLET, 0.5 mg/kg) has no effect on the incidence rate of ischemic and reperfusion arrhythmias. Preliminary administration of glibenclamide (0.3 mg/kg), an inhibitor of K_ATP channels, blocks the antiarrhythmic effect of DALDA and DPDPE. Repeated short-term exposures of rats to immobilization within two weeks increases the heart tolerance to the arrhythmogenic effect of coronary occlusion and reperfusion. This effect disappears after administration of CTAP (0.5 mg/kg), a antagonist, or injection of 5-hydroxydecanoate (5 mg/kg), an inhibitor of mitochondrial K_ATP channels. The selective antagonists of and ORs have no effect on cardiac adaptation-induced resistance to the arrhythmogenic effect of ischemia and reperfusion. We believe that stimulation of , , and ORs increases myocardial tolerance to the arrhythmogenic effect of ischemia and reperfusion through activation of K_ATP channels. The antiarrhythmic effect of the adaptation is mediated by stimulation of ORs and mitochondrial K_ATP channels.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

The Opioidergic System in the Combined Regulation of Pain and Immunity

Opioidergic mechanisms are involved in responses to nociceptive and antigenic stimuli at all levels and stages (from peripheral nociceptors to the cerebral cortex and from the precursors of immunocompetent cells to mature effector cells). In most experimental and clinical studies, opioid-mediated analgesia proved to be accompanied by immunosuppression. Opioid receptors of , , and types are involved in the mechanisms of combined regulation of pain and immunity, with and receptors suppressing the immune response and receptors enhancing it. By modifying the chemical structure of opioid ligands, it is possible to preserve the analgesic effect and avoid the development of immunosuppression. The opioidergic mechanisms are coupled with nonopioid peptidergic and nonpeptide systems of pain and immunity regulation.     

Expression of a bovine κ-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine -casein (-CN) cDNA under the control of the goat -CN 5 promoter elements and 3 flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine -CN RNA to the mammary gland and secretion of bovine -CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine -CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69% and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine -CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine -CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did -CN from bovine milk. A high degree of variation in the production of bovine -CN within each of the transgenic lines was observed.     

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

Summary With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible (Red River), one tolerant (UC 105) and three resistant (Davis UC 82, Heinz, UC 90) tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo — resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.     

Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-B activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85 subunit of PI3-kinase interacted with tyrosine-phosphorylated IB- in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-B activation. The inhibition of NF-B activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-B p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-B activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-B activation through interaction with tyrosine-phosphorylated IB- and contributing to tyrosine phosphorylation of p65 NF-B.     

Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue

Summary An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a direct fluorescence procedure using FTC-toxin, but not by an indirect procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the direct and indirect procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the indirect procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a direct procedure using FTC-antiprofilaggrin but only weakly by an indirect double antibody procedure. Insensitivity to indirect procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.