Immunofluorescence assay for the quantitative and qualitative evaluation of intracellular interleukin-8 in microtiter plates.

In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular protein (colorimetric assay). The quantitative and qualitative determination of intracellular IL-8 was achieved by immunofluorescence using the ELF-detection system (Molecular Probes, Eugene, OR), which is approximately 10 times more sensitive than conventional immunofluorescence detection systems. Thus, a 32x objective magnification (without immersion oil) is sufficient to precisely assess the subcellular localization of IL-8.

Real-Time Monitoring of the Catalytic Oxidation of Alcohols to Aldehydes and Ketones on Resin Support by Single-Bead Fourier Transform Infrared Microspectroscopy.

Catalytic oxidations of primary, benzylic, and secondary alcohols to aldehydes and ketone using tetra-n-propylammonium perruthenate (TPAP) were carried out on resin supports for the first time. The reaction time course, percent conversion, and influence of catalyst amount have been determined by analyzing IR spectra taken directly on a single resin bead in real time. Using 0.

Membrane-bound ezrin is involved in B-cell receptor-mediated signaling: potential role of an ITAM-like ezrin motif.

Ezrin is a cytoskeleton-plasma membrane linker molecule which is implicated in the T-cell antigen receptor signaling as one of the major tyrosine phosphorylated components. Its function in B-lymphocyte activation has not yet been clarified. Here we studied the potential involvement of ezrin in the B-cell receptor (BCR) signaling in BL41 Burkitt lymphoma cells.

VCAM-1/alpha 4-integrin adhesion pathway: therapeutic target for allergic inflammatory disorders.

Lymphocyte recirculation and leukocyte extravasation involve a multistep process that is central to immune surveillance and the rapid response of white blood cells to sites of injury or infection. Interaction of vascular adhesion molecules (VCAM-1, ICAM-1, and selectins) with ligands on the leukocyte surface (integrins, carbohydrates, and mucin-like molecules) regulate diapedesis. The nature of an inflammatory stimulus ultimately determines the pattern of endothelial adhesion molecule expression and the avidity state of their counterreceptors, thus dictating to a large extent whether a subclass of leukocytes will play a dominant role in the immune response.

Induction of cognate and non-cognate T-cell help for B-cell IgE production in relation to CD40 ligand expression.

Nonactivated, fixed peripheral blood T cells (PBT) from healthy donors or patients with X-linked-hyper-IgM (HIGM) syndrome, or cloned T cells provided effective help for tonsillar B lymphocytes for induction of IgE or other immunoglobulin (Ig) isotypes. Helper activity was mediated by staphylococcal superantigens adsorbed to the T cells prior to fixation and required presence of IL-4 in the cultures. We demonstrated that the T cells neither expressed detectable CD40 ligand at the beginning of the superantigen treatment nor 24 h later.

Extracts from rhizomes of Cyperus articulatus (Cyperaceae) displace [3H]CGP39653 and [3H]glycine binding from cortical membranes and selectively inhibit NMDA receptor-mediated neurotransmission.

The marshland plant Cyperus articulatus (Cyperaceae) is commonly used in traditional medicine in Africa and Latin America to treat a wide variety of human diseases ranging from headache to epilepsy. We tested the hypothesis that the purported anti-epileptic effect of this plant might be due to a functional inhibition of excitatory amino acid receptors. One or several component(s) contained in the extracts inhibited the binding of [3H]CGP39653 to the NMDA recognition site and of [3H]glycine to the strychnine-insensitive glycine site of the NMDA receptor complex from rat neocortex.

Amyloid precursor protein truncated at any of the gamma-secretase sites is not cleaved to beta-amyloid.

beta A4 secretion occurs upon processing of amyloid protein precursor (APP) by beta-secretase (N-terminus of beta A4) and gamma-secretase (C-terminus). To determine the sequence of these activities and the processing intermediate of beta A4, we expressed several truncated APP molecules in human HEK-293 cells. Immunofluorescence and biotinylation studies indicated that full-length APP or APP lacking the cytosolic domain both were located intracellularly, associated with the cell surface and secreted.

The competitive NMDA receptor antagonist SDZ 220-581 reverses haloperidol-induced catalepsy in rats.

The present studies investigated whether SDZ 220-581 ((S)-alpha-amino 2'chloro-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid), a potent, competitive antagonist at the NMDA glutamate receptor subtype, reversed haloperidol-induced catalepsy in rats, a widely used model of Parkinson's disease. SDZ 220-581 (0.32-3.

Synergistic activation of interleukin-8 gene transcription by all-trans-retinoic acid and tumor necrosis factor-alpha involves the transcription factor NF-kappaB.

Induction of interleukin-8 (IL-8) by IL-1 or tumor necrosis factor (TNF), and repression by interferons or glucocorticoids have been shown to involve sequences between nucleotides -94 and -71 of the 5'-flanking region, and the transcription factors NF-IL-6 and NF-kappaB. The A3 cell line was derived from the human melanoma cell line G-361 by stable transfection with part of the IL-8 promoter (nucleotides -101 to +40 from transcription start) fused to the luciferase coding region. These regulatory sequences were sufficient for transcriptional activation by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, IL-1beta, or TNF-alpha.

Progression of Organic Reactions on Resin Supports Monitored by Single Bead FTIR Microspectroscopy.

We report the time courses of five solid-phase reactions obtained using single bead FTIR microspectroscopy. This time-resolved information aided in the determination of the required reaction time, the nature of the solid-phase reaction, and resin property, effectively assisting in the initial phase of our combinatorial chemistry efforts. Our results showed that solid-phase organic reactions proceed faster than generally speculated.

Induction of rapid IL-1 beta mRNA degradation in THP-1 cells mediated through the AU-rich region in the 3'UTR by a radicicol analogue.

A radicicol analogue (analogue A) was found to inhibit interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) secretion from THP-1 cells. If added to cells activated by interferon gamma and lipopolysaccharide, radicicol analogue A not only inhibited the secretion of IL-1 beta but also induced an extremely rapid degradation of IL-1 beta, IL-6 and TNF-alpha mRNA to undetectable levels within 5-8 h. This degradation is independent of translation and of the signal inducing transcription.

The pharmacokinetics and bioavailability of clemastine and phenylpropanolamine in single-component and combination formulations.

Studies were conducted in healthy male volunteers (n = 171; age range, 19-49 years; 22-27 subjects per study) to examine the following: pharmacokinetics and dose proportionality of the antihistamine clemastine; the effect of coadministration of phenylpropanolamine and clemastine on the pharmacokinetics of the two drugs; and the bioavailability of clemastine tablets and combination tablets of clemastine and sustained-release phenyl-propanolamine under fasted and fed conditions after single-dose administration and at steady state. All studies used crossover designs, with randomized drug treatments separated by a 7-day washout period for the single-dose studies, and with administration every 6 or 12 hours for 7 days per treatment for the steady-state studies. After single oral doses of clemastine solution (1,2, and 4 mg), the area under the concentration-time curve (AUC) and maximum concentration (Cmax) were dose proportional.

High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization.

Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis.

Induction of the TNF-alpha promoter in the murine dendritic cell line 18 and the murine mast cell line CPII is differently regulated.

While it was recently shown that activation of dendritic cells (DC) results in the production of a number of cytokines, the signal pathways and transcription factors involved in this process have not been described. To address this issue we compared the events resulting in the activation of the human TNF-alpha promoter occurring in the fetal dendritic cell line 18 (DC18) with those in the well-characterized murine mast cell line CPII. As stimuli we employed the protein kinase C inducer, PMA, and the Ca2+ ionophore, ionomycin, both of which are known to activate a large variety of intracellular signaling pathways.

Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450-specific chemical inhibitors on this activation in human liver microsomes.

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-methyl and alpha-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (alpha-methyl hydroxylation), and keto aldehyde (alpha-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus-insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the alpha-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol.

Kinetics and isotype profile of rheumatoid factor production during viral infection: organ distribution of antibody secreting cells.

The kinetics and isotype profile of influenza virus-specific IgG antibodies were studied in correlation with the serum titre of IgG-reactive autoantibodies. An increased level of IgG isotype-specific, rheumatoid factor-type autoantibody secretion was observed in the late phase of the virus-specific memory response. These rheumatoid factors were specific for the IgG2a and IgG1 subclasses which dominated the anti-viral antibody response.

Methods for Study of Chemokine Receptors in the Tissues.

Three different assays were used to study the distribution of binding sites for IL-8 in human skin and several animal tissues. An in situ binding assay was designed in which the binding of radiolabeled IL-8 to small intact tissue pieces was studied, and a histological autoradiographic technique was used to detect the bound chemokine in the subsequently prepared tissue sections. A modified assay was also performed in which the binding of unlabeled IL-8 to intact tissue pieces was visualized using monoclonal anti-IL-8 antibody.

Errors in time in pharmacokinetic studies.

Pharmacokinetic models with errors in the time variable were developed and explored by simulation in NONMEM. The precision of estimated parameters decreased with increasing error magnitude. The usual asymptotic standard error estimates were biased low by 10-50%.

Serial versus sparse sampling in toxicokinetic studies.

Purpose: Sparse sampling in rodent toxicokinetics usually involves the collection of a single blood sample on a given study day from each animal in a treatment group. The samples are allocated to different time points, often allowing some replicates, and statistical inferences are then made about the concentration-time behavior of the test compound. The present study compared the results of one such analysis with those obtained from serial sampling as might be applied using satellite animals.

Evidence for an alpha helical T cell epitope in the C-terminus of the main birch pollen allergen Bet V 1.

Secondary structure prediction of the main birch pollen allergen Bet v 1 was found to be in good agreement with the secondary structural elements found by analysing the Bet v 1 circular dichroism data. According to both experiment and prediction, 32% of 160 amino acids participate in alpha helices, 21% in beta sheets, 24% in turns, and 23% in other structural motifs. The peptide LRAVESYLLAHS which represents one of the major T cell epitopes on Bet v 1 was shown to have a high propensity to form an alpha helix.