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Prolonged Incubation (prolonged + incubation)
Selected AbstractsThe proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complexFEBS JOURNAL, Issue 17 2004Joanne L. Cowan The eukaryotic translation initiation factor (eIF) 4E, is regulated by modulating both its phosphorylation and its availability to interact with the scaffold protein, eIF4G, to form the mature eIF4F complex. Here we show that treatment of C2C12 myoblasts with the proteasomal inhibitor, MG132 (N -carbobenzoxyl-Leu-Leu-leucinal), resulted in an early decrease in protein synthesis rates followed by a partial recovery, reflecting the reprogramming of translation. The early inhibition of protein synthesis was preceded by a transient increase in eIF2, phosphorylation, followed by a sustained increase in eIF4E phosphorylation. Inhibition of eIF4E phosphorylation with CGP57380 failed to prevent translational reprogramming or the moderate decrease in eIF4F complexes at later times. Prolonged incubation with MG132 resulted in the increased expression of heat shock protein (hsp)25, ,B-crystallin and hsp70, with a population of hsp25 associating with the eIF4F complex in a p38 mitogen-activated protein kinase-dependent manner. Under these conditions, eIF4GI, and to a lesser extent eIF4E, re-localized from a predominantly cytoplasmic distribution to a more perinuclear and granular staining. Although MG132 had little effect on the colocalization of eIF4E and eIF4GI, it promoted the SB203580-sensitive association of eIF4GI and hsp25, an effect not observed with ,B-crystallin. Addition of recombinant hsp25 to an in vitro translation assay resulted in stimulation of on-going translation and a moderate decrease in de novo translation, indicating that this modified eIF4F complex containing hsp25 has a role to play in recovery of mRNA translation following cellular stress. [source] Caldesmon is a cytoskeletal target for PKC in endotheliumJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2006Natalia V. Bogatcheva Abstract We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of PMA-induced actin and myosin redistribution is not followed by caldesmon redistribution. Co-immunoprecipitation experiments revealed that short-term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15,60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing PMA-induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA-induced cytoskeletal rearrangement. Our results suggest that PKC-dependent phosphorylation of caldesmon is involved in PMA-mediated complex cytoskeletal changes leading to the EC barrier compromise. J. Cell. Biochem. 99: 1593,1605, 2006. © 2006 Wiley-Liss, Inc. [source] IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARPJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004MIN-JIE CAO ABSTRACT Myofibril-bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55,60C as shown by SDS-PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as ,-actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril-bound serine proteinase. [source] Patterns of Interaction between Populus Esch5 and Piriformospora indica: A Transition from Mutualism to AntagonismPLANT BIOLOGY, Issue 2 2005M. Kaldorf Abstract: Piriformospora indica (Sebacinaceae, Basidiomycota) is an axenically cultivable, plant growth promoting root endophyte with a wide host range, including Populus. Rooting of Populus Esch5 explants started within 6 days after transfer to WPM medium. If such plantlets with roots were inoculated with P. indica, there was an increase in root biomass, and the number of 2nd order roots was increased significantly. A totally different observation was recorded when the explants were placed into WPM with pre-grown P. indica. The interaction led to complete blocking of root production and severely inhibited plant growth. Additionally, branched aerial roots appeared which did not penetrate the medium. On contact with the fungal colony or the medium, the ends of the aerial roots became inflated. Prolonged incubation stimulated the fungus to colonize aerial parts of the plant (stem and leaves). Mycelium not only spread on the surface of the aerial parts, but also invaded the cortical tissues inter- and intracellularly. Detached Populus leaves remained vital for 4 - 5 weeks on sterile agar media or on AspM medium with pre-grown P. indica. When the fungus was pre-grown on culture media such as WPM, containing ammonium as the main source of nitrogen, leaves in contact with the cultures turned brownish within 4 - 12 h. Thereafter, the leaves bleached, and about one day later had become whitish. Thus, cultural conditions could alter the behaviour of the fungus drastically: the outcome of the interaction between plant and fungus can be directed from mutualistic to antagonistic, characterized by fungal toxin formation and extension of the colonization to Populus shoots. [source] Full-length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathwaysFEBS JOURNAL, Issue 9 2008Driss El Moustaine As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes ,-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, AggHP, which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, AggHP transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially ,-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, IHP, is formed under high pressure. After pressure release, IHP relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, IHP, constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids. [source] Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395FEBS JOURNAL, Issue 12 2004Adrian J. Dunford In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (e.g. methionine synthase reductase and novel reductase 1), and tyrosine in plant ferredoxin-NADP+ reductase. Trp676 in human cytochrome P450 reductase is conformationally mobile, and plays a key role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase. Herein, we describe studies of rat neuronal nitric oxide synthase FAD domains, in which the aromatic shielding residue Phe1395 is replaced by tryptophan, alanine and serine. In steady-state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wild-type. Stopped-flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains, suggesting that this contributes to altered preference in coenzyme specificity. Unlike cytochrome P450 reductase, the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH, but probably results from improved geometry for hydride transfer in the F1395S, and F1395A,NADH complexes. Potentiometry indicates that the substitutions do not significantly perturb thermodynamic properties of the FAD, although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S, consistent with changes in hydrophobicity of the flavin environment. In wild-type and F1395W FAD domains, prolonged incubation with NADPH results in development of the neutral blue semiquinone FAD species. This reaction is suppressed in the mutant FAD domains lacking the shielding aromatic residue. [source] Enhanced exoenzyme activities in sediments in the presence of deposit-feeding Chironomus riparius larvaeFRESHWATER BIOLOGY, Issue 9 2007PETER STIEFArticle first published online: 10 JUN 200 Summary 1. The combined effects of deposit-feeding, bioturbation and bioirrigation by benthic macrofauna on the enzymatic hydrolysis of organic matter were studied in microcosms. Chironomus riparius larvae (Insecta, Diptera) served as model macrofauna and stinging nettle leaves (Urtica dioica) were used as a detrital food source. 2. In the upper 10 mm of the sediment (the habitat of C. riparius larvae), the activities of several exoenzymes, the contents of several fractions of particulate organic matter (POM), and the concentrations of dissolved oxidants (O2, NO) were measured on a small scale. Fluorescent particles (luminophores) were used to quantify the vertical redistribution of particles within the same layer. 3. In control sediment, the addition of detrital food enhanced exoenzyme activities in the 0,2 mm layer only. In the presence of C. riparius larvae, exoenzyme activities increased to 10 mm depth. Further, the content of POM in the 0,2 mm layer was lower in the presence than in the absence of larvae, suggesting ingestion and subduction of the added detritus. After prolonged incubation without further food addition, exoenzyme activities returned close to background values in both treatments, whereas the vertical distribution of POM remained unchanged. 4. The overall penetration depth of O2 and NO into the sediment was greater in the presence than the absence of C. riparius, the differences being more pronounced after prolonged incubation. Locally high O2 and NO concentrations due to bioirrigation by C. riparius were measured deep in the sediment. Net downward transport of particles was observed only in the presence of C. riparius larvae and only at the beginning of the incubation. 5. I conclude that deposit-feeding and bioturbation by macrofauna can quickly remove freshly deposited POM from the sediment surface and transfer it to less oxygenated sites (i.e. animal guts and deep sediment layers). Bioirrigation also increases the availability of oxidants deep in the sediment. The oscillation of oxidant supply to POM particles by ingestion,egestion, burial and re-burial, and the intermittent bioirrigation of subsurface sediment, is probably the cause of the increased rate of organic matter hydrolysis, the rate-limiting step in mineralization. [source] Granulocyte,macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanismsIMMUNOLOGY, Issue 2 2000A. Bergamini Summary Granulocyte,macrophage colony-stimulating factor (GM-CSF) has multiple effects on the antigen phenotype and function of macrophages. In this study we investigated the effect of GM-CSF on cytokine production by macrophages. We found that GM-CSF may modify the tumour necrosis factor-, (TNF-,) and interleukin-6 (IL-6) response to lipopolysaccharide (LPS) through two different mechanisms. Relatively early in culture, GM-CSF increases the amount of cytokines synthesized by responding cells; this effect appears to be unrelated to modulation of CD14 expression and LPS-binding capacity. After prolonged incubation, GM-CSF up-regulates both CD14 expression and LPS-binding capacity, and the frequency of cytokine-producing cells. Release of CD14 in the culture supernatant was decreased in the presence of GM-CSF, suggesting that a reduced shedding was responsible for the effect of GM-CSF on CD14 expression. Enhancement of cytokine production was also observed in GM-CSF-treated macrophages after stimulation by phorbol 12-myristate 13-acetate (PMA), thus indicating that GM-CSF affects both CD14-dependent and -independent cytokine production. Finally, GM-CSF did not modulate the LPS- and PMA-induced production of IL-10 and IL-12. We conclude that GM-CSF may play a role in manipulating the activation-induced expression of pro-inflammatory cytokines by macrophages. Enhanced production of these cytokines could play an important role in the pathogenesis of Gram-negative septic shock syndrome and in defence against infectious agents. [source] Ex vivo expansion of apheresis-derived peripheral blood hematopoietic progenitorsJOURNAL OF CLINICAL APHERESIS, Issue 1 2002Zeev Estrov Because the administration of hematopoietic growth factors and the use of stem cell support often fails to alleviate the neutropenic phase induced by cytotoxic drugs, several investigators have attempted to expand ex vivo hematopoietic progenitors for clinical use. These attempts have clearly shown that the cultured cells are functional and can be safely administered to patients, but that the in vivo performance is disappointing and the concept as a whole is not yet clinically useful. The major reasons for these unsuccessful attempts are thought to be cumbersome cell fractionation techniques, contamination, prolonged incubation, and the use of less than ideal cytokine combinations. In response, we have developed a simple procedure for ex vivo expansion of myeloid progenitor cells. In this assay, unfractionated mononuclear cells from apheresis donors are incubated in nonpyrogenic plastic bags for 7 days in the presence of culture medium either containing fetal calf serum or human plasma, granulocyte colony-stimulating factor, and stem cell factor. We have demonstrated that under these conditions the number of colony-forming units (CFU) granulocyte-macrophage (CFU-GM) and of CFU-granulocyte-macrophage-erythroid-megakaryocyte (CFU-GEMM) increased 7- and 9-fold, respectively, by day 7 and the number of burst-forming units-erythroid (BFU-E) increased 2.7-fold by day 5 of culture. Significant increases in the numbers of cells expressing CD34+, CD34+/CD38+, CD34+/CD33+, CD34+/CD15+, and CD34+/CD90+ and significant declines in the numbers of cells expressing CD34+/CD38- and CD19 surface antigens were also observed. The relative numbers of cells expressing T-cell markers and CD56 surface antigen did not change. By using different concentrations of various hematopoietic growth factor combinations, we can increase the number of mature and immature cells of different hematopoietic lineages. J. Clin. Apheresis 17:7,16, 2002. © 2002 Wiley-Liss, Inc. [source] The [Tc(N)(PNP)]2+ metal fragment labeled cholecystokinin-8 (CCK8) peptide for CCK-2 receptors imaging: in vitro and in vivo studiesJOURNAL OF PEPTIDE SCIENCE, Issue 4 2007Stefania Agostini Abstract The radiolabeling of the natural octapeptide CCK8, derivatized with a cysteine residue (Cys-Gly-CCK8), by using the metal fragment [99mTc(N)(PNP3)]2+ (PNP3 = N,N -bis(dimethoxypropylphosphinoethyl)methoxyethylamine) is reported. The [99mTc(N)(NS-Cys-Gly-CCK8)(PNP3)]+ complex was obtained according to two methods (one-step or two-step procedure) that gave the desired compound in high yield. The complex is stable in aqueous solution and in phosphate buffer. In vitro challenge experiments with an excess of cysteine and glutathione indicate that no transchelation reactions occur, confirming the high thermodynamic stability and kinetic inertness of this compound. Stability studies carried out in human and mouse serum, as well as in mouse liver homogenates, show that the radiolabeled compound remains intact for prolonged incubation at 37 °C. Binding properties give Kd (19.0 ± 4.6 nmol/l) and Bmax (,106 sites/cell) values in A431 cells overexpressing the CCK2-R. In vivo evaluation of the compound shows rapid and specific targeting to CCK2-R, a fourfold higher accumulation compared to nonreceptor expressing tumors. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Synthesis and evaluation of ampicillin-conjugated gum arabic microspheres for sustained releaseJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2007K. K. Nishi Ampicillin was conjugated to periodate-oxidized gum arabic (GA), a branched polysaccharide, to form the imino conjugate of the drug and the polysaccharide. The water-soluble conjugate was dispersed by sonication in a mixture of toluene and liquid paraffin in the presence of a non-ionic surfactant as droplet stabilizer and fabricated into microspheres by heat denaturation at 80°C to obtain spheres less than 2 ,m in diameter. These microspheres did not undergo dissolution in water on prolonged incubation. In-vitro release of ampicillin into phosphate buffer from the microspheres was slow and sustained with a cumulative release between 10 and 25% of the drug content in 10 days depending on the degree of oxidation of GA and the drug payload. Release into simulated gastric fluid was faster due to faster hydrolysis of the drug-GA bond in the acid medium, but when the medium was changed to intestinal fluid, the release was slowed down. Ampicillin released was functionally active and inhibited the growth of S. aureus and E. coli in cultures, although not as actively as free ampicillin. The microspheres underwent slow biodegradation on prolonged incubation in aqueous media. These studies show that ampicillin conjugated with oxidized GA and fabricated into microspheres possesses sustained-release characteristics for prolonged periods. [source] Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobioseMOLECULAR MICROBIOLOGY, Issue 6 2007Aashiq H. Kachroo Summary Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiose-positive (Cel+) mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. We show that the Cel+ derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of N,N,-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into single-amino-acid substitutions. Introduction of chbR from two Cel+ mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a Cel+ phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and phospho-,-glucosidase, upon induction, could recognize, transport and cleave cellobiose respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-,-glucosidase under a heterologous promoter. Biochemical characterization of one of the chbR mutants, chbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of cellobiose 6-phosphate as an inducer compared with the wild-type regulator. [source] Growth phase-dependent expression and degradation of histones in the thermophilic archaeon Thermococcus zilligiiMOLECULAR MICROBIOLOGY, Issue 4 2000Marcel E. Dinger HTz is a member of the archaeal histone family. The archaeal histones have primary sequences and structural similarity to the eukaryal histone fold domain, and are thought to resemble the archetypal ancestor of the eukaryal nucleosome core histones. The effects of growth phase on the total soluble proteins from Thermococcus zilligii, isolated after various stages of growth from mid-logarithmic to late stationary phase, were examined by denaturing polyacrylamide gel electrophoresis. On entry into stationary phase, at least 11 proteins were detected that changed considerably in level. One of these proteins was identified by Western hybridization as HTz. The level of HTz decreased dramatically as cells entered stationary phase, and it could not be detected by late stationary phase. Unexpectedly, the Western hybridization detected a second protein, with an estimated molecular mass of approximately 14 kDa, which paralleled the decrease in level of HTz. Native purified HTz was shown to retain complete activity after prolonged incubation at the growth temperature of the organism, suggesting that the decrease in HTz was a specific cell-regulated process. Analysis of native purified HTz by electrospray ionization mass spectrometry revealed the molecular masses of HTz1 and HTz2 to be 7204 ± 3 Da and 7016 ± 3 Da respectively. The only non-covalent species that was detected corresponded to the molecular mass of an HTz1,HTz2 heterodimer. Northern analyses of T. zilligii total RNA with an htz1 gene probe indicated a rapid decrease in expression of htz1 with progression of the growth phase, and complete repression of htz1 transcript synthesis by late logarithmic phase. Three proteins that changed in level with growth phase were identified by N-terminal sequence analysis. The first was homologous to a hypothetical protein conserved in all Archaea sequenced to date, the second to the Sac10b family of archaeal DNA-binding proteins and the third to the C-terminal region of the leucine-responsive regulatory family of DNA-binding proteins (LRPs). [source] Proteome analysis of the phenotypic variation process in Photorhabdus luminescensPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2006Evelyne Turlin Dr. Abstract Photorhabdus luminescens is an insect pathogen associated with specific soil nematodes. The bacterium has a complex life cycle with a symbiotic stage in which bacteria colonize the intestinal tract of the nematodes, and a pathogenic stage against susceptible larval-stage insect. Symbiosis-"deficient" phenotypic variants (known as secondary forms) arise during prolonged incubation. Correspondence analysis of the in silico proteome translated from the genome sequence of strain TT01 identified two major biases in the amino acid composition of the proteins. We analyzed the proteome, separating three classes of extracts: cellular, extracellular, and membrane-associated proteins, resolved by 2-DE. Approximately 450 spots matching the translation products of 231 different coding DNA sequences were identified by PMF. A comparative analysis was performed to characterize the protein content of both variants. Differences were evident during stationary growth phase. Very few proteins were found in variant II supernatants, and numerous proteins were lacking in the membrane-associated fraction. Proteins up-regulated by the phenotypic variation phenomenon were involved in oxidative stress, energy metabolism, and translation. The transport and binding of iron, sugars and amino acids were also affected and molecular chaperones were strongly down-regulated. A potential role for H-NS in phenotypic variation control is discussed. [source] A Recombinant Bacteriophage-Based Assay for the Discriminative Detection of Culturable and Viable but Nonculturable Escherichia coli O157:H7BIOTECHNOLOGY PROGRESS, Issue 3 2006Raheela Awais A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e - /GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation ofE. coli O157:H7 cells with PP01e - /GFP did not lead to cell lysis, while the propagation of PP01e - /GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e - /GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count. [source] Expression of the neutrophil-activating CXC chemokine ENA-78/CXCL5 by human eosinophilsCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2003T. Persson Summary Background Eosinophils are seen at sites of inflammation in diseases such as helminthic infestation, asthma, ulcerative colitis and some neoplastic diseases. They are also associated with connective tissue remodelling, for example in longstanding asthma. In the present study, we investigated whether eosinophils express the CXC chemokine epithelial cell-derived neutrophil activating peptide (ENA-78/CXCL5), a chemokine that can activate neutrophils and in addition possesses angiogenic properties. Immunocytochemistry detected CXCL5 in eosinophils and the peptide was localized in the specific granules by immunoelectron microscopy. Methods and Results In eosinophil lysates, 12 ± 2 pg (mean ± SEM) of CXCL5 was detected per 106 cells by enzyme-linked immunosorbent assay (ELISA). Weak constitutive expression of CXCL5, as well as the related CXC chemokine IL,8/CXCL8, could be detected in freshly isolated eosinophils by RT,PCR. However, during prolonged incubation of eosinophils, a strong increase in both CXCL5 and IL-8/CXCL8 expression was seen, as detected by RT-PCR, and increasing amounts of CXCL5 peptide with time were detected in the incubation medium by ELISA. Addition of TNF-, neutralizing antibodies during prolonged incubation significantly inhibited CXCL5 production, demonstrating involvement of auto- and paracrine effects from TNF-, produced by eosinophils themselves. Addition of IFN-, showed a strong inhibitory effect on CXCL5 synthesis. Conclusion These findings suggest that, through expression of CXCL5, eosinophils can recruit and activate CXC receptor 2 (CXCR2)-bearing cells such as neutrophils at sites of inflammation. Eosinophils may also promote connective tissue remodelling through release of this peptide. [source] Enhancement of Th2 pathways and direct activation of B cells by the gingipains of Porphyromonas gingivalisCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2003L. W. P. YUN SUMMARY Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process that has been linked with Th2 pathways. Critical in maintaining Th2 activity is the response of B lymphocytes to environmental interleukin (IL)-4, a cytokine that also counteracts Th1-cell differentiation. Here we demonstrate that while the gingipains effectively degrade interleukin (IL)-4 under serum-free conditions, limited hydrolysis was observed in the presence of serum even after prolonged incubation. Gingipains up-regulated CD69 expression directly in purified peripheral blood B cell preparations. Further, the induction of IL-4 receptor expression on B cells by gingipains correlates with B cell activation, which is also manifested by a mitogenic response. These results suggest that the gingipains of P. gingivalis act during the early stage of B-cell growth as a competence signal, whereby sensitized B cells might become more responsive to further challenge in the disease-susceptible individual. [source] | ||||||||||||||||