Effects of the 5-lipoxygenase inhibitors AA-863 and U-60,257 on human glioma cell lines

The effects of two specific 5-lipoxygenase inhibitors AA-863 and U-60,257 (piriprost) on the growth of two human glioma cell lines, U-343 MGa and U-251 MG were investigated. Both monolayer cultured cells and spheroids were studied. The results of the monolayer studies showed potent and dose dependent inhibitory effects on the proliferation of glioma cells (IC50/one week treatment/of AA-863: 9.0 microM, IC50 of U-60,257: 40.0 microM). The experiments made on the tumor spheroids suggested an inhibitory effect on proliferation and volume growth already at lower doses (AA-863: 0.4-2.0 microM, U-60,257: 1.0-5.0 microM), a dose range where effects were not found in monolayers. At higher doses (AA-863: 10.0-30.0 microM, U-60,257: 30.0-90.0 microM) the experiments with spheroids failed to demonstrate a further inhibitory effect on spheroid volume, probably attributed to phenomena such as swelling of cells, dissociation of spheroid structure and development of necrosis. The clearly dose dependent inhibitory effect on the proliferation of human glioma cells in monolayer culture and the inhibitory effects on spheroid growth with these specific inhibitors indicate a role for lipoxygenase products in the growth of gliomas.     

Cardiovascular and Pulmonary Effects of a New Sedative/Analgesic (Medetomidine) as a Preanaesthetic Drug in the Dog

Cardiovascular and pulmonary effects of a new sedative/analgesic (medetomidine) as a preanaesthetic drug in the dog. A study was carried out to investigate the possible usefulness of medetomidine (Farmos Group, Turku, Finland) for premedication prior to general anaesthesia with thiopental sodium and halothane. The main emphasis was laid on the circulatory and respiratory effects of medetomidine. Dogs treated with xylazine (2 mg/kg) or placebo (physiological saline solution) served as controls. Medetomidine caused a decrease in blood pressure, heart rate and respiratory rate at all dose levels tested. These decreases were essentially dose -dependent, but there were great individual variations. It is concluded that the drug can be useful for premedication at the lowest dose level tested (10 μ/kg). The sedative effect, however, is so strong that an even lower dose might be sufficient for the present purpose.     

On disturbance of homeostasis in organ-cultured tissue

Summary The intact membranous rat mesentery was cultured in Eagle's minimum essential medium containing no serum or only low concentrations of serum. The procedure is in some important respects superior to previous organ culture techniques. To estimate the extent of disturbance of homeostasis of the tissue in culture, the spontaneous mast-cell histamine release was quantitated after preculture preparation of the specimens and after different intervals in culture. Also, the proliferation of fibroblasts and mesothelial cells that predominate in the mesentery was assessed at 48 h by cytofluorometric quantitation of DNA in single-tissue cells. Spontaneous histamine release was time dependent during cultivation, amounting to ca. 50% at 48 h, and was affected by the medium used for moistening the tissue before cultivation. Culturing also brought about great spontaneous increase in the proliferation of fibroblasts and mesothelial cells, the rate being related to the concentration of serum. Addition of the mast-cell secretagogues 48/80 or polymyxin B at 1 h caused rapid release of 50 to 60% of the histamine and was followed by augmented proliferation in the serum-containing media. The spontaneous increase of cell proliferation in tissue culture may be causally related to mast-cell secretion. Further studies are needed to define factors influencing the spontaneous mast-cell secretion and the mast-cell-dependent mitogenesis in normal tissue cells Supported by grants from the Swedish Medical Research Council (Project 5942) and State Board for Animal Experiments.     

Escherichia coli K-12 mutants hyperproducing chromosomal beta-lactamase by gene repetitions.

Escherichia coli K-12 ampicillin-resistant mutants hyperproducing chromosomal beta-lactamase arose spontaneously from strains carrying ampA1 ampC(+). Such mutants were found even in a recA background. Two Amp(r)-100 strains were analyzed genetically. The Amp(r)-100 resistance level of both strains could be transduced by direct selection for ampicillin resistance. Several classes of ampicillin-resistant transductants were found that differed from one another in the beta-lactamase activity and the ampicillin resistance mediated by an ampA1 ampC(+)-carrying strain. The data suggested that beta-lactamase hyperproduction was due to repetitions of the chromosomal amp genes. The size of the repeated region was calculated from cotransduction estimates, using the formula of Wu (Genetics 54:405-410, 1966), and was found to be about 1 min in one strain and 1.5 min in the other. Second-step Amp(r)-400 mutants were isolated from an Amp(r)-100 strain. The resistance of these mutants was apparently also due to repetitions, each mediating a resistance to about 10 mug/ml. Mutants of wild-type strains that were moderately resistant to ampicillin also gave rise to intermediate-resistance classes, suggesting repetitions of the wild-type amp alleles. F' factors hyperproducing chromosomal beta-lactamase by gene repetitions were constructed. They mediated levels of ampicillin resistance comparable to that of naturally occurring resistance plasmids. The expression of beta-lactamase hyperproduction was not affected by the presence of ampA and ampC alleles in trans and did not act in trans on the other alleles.     

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.     

Borrelia pathogenesis research in the post-genomic and post-vaccine era

In the two years after publication of the genome sequence of Borrelia burgdorferi and reports on human field trials of a vaccine against Lyme borreliosis, there has been further progress in understanding of host-parasite interactions during Lyme borreliosis and relapsing fever. Some mechanisms that Borrelia spirochetes use to avoid elimination and to persist in the host are novel. In addition, the recent discovery of antigenic variation in the Lyme disease agent B. burgdorferi adds to the complexity of the possible virulence properties of this human pathogen.     

High-Resolution,Non-Invasive Imaging of Upper Vocal Tract Articulators Compatible with Human Brain Recordings

A complete neurobiological understanding of speech motor control requires determination of the relationship between simultaneously recorded neural activity and the kinematics of the lips, jaw, tongue, and larynx. Many speech articulators are internal to the vocal tract, and therefore simultaneously tracking the kinematics of all articulators is nontrivial—especially in the context of human electrophysiology recordings. Here, we describe a noninvasive, multi-modal imaging system to monitor vocal tract kinematics, demonstrate this system in six speakers during production of nine American English vowels, and provide new analysis of such data. Classification and regression analysis revealed considerable variability in the articulator-to-acoustic relationship across speakers. Non-negative matrix factorization extracted basis sets capturing vocal tract shapes allowing for higher vowel classification accuracy than traditional methods. Statistical speech synthesis generated speech from vocal tract measurements, and we demonstrate perceptual identification. We demonstrate the capacity to predict lip kinematics from ventral sensorimotor cortical activity. These results demonstrate a multi-modal system to non-invasively monitor articulator kinematics during speech production, describe novel analytic methods for relating kinematic data to speech acoustics, and provide the first decoding of speech kinematics from electrocorticography. These advances will be critical for understanding the cortical basis of speech production and the creation of vocal prosthetics.     

Design,synthesis and structure–activity relationships of zwitterionic spirocyclic compounds as potent CCR1 antagonists

A series of zwitterionic spirocyclic compounds were synthesised. In vitro data revealed that these compounds were potent CCR1 antagonists. In particular, 2, 4, 11 and 20 inhibited CCR1 mediated chemotaxis of THP-1 cells in a functional assay.     

Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties

The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80-104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55-69 and aa 80-104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody-assisted epitope analysis of N-acetylgalactosaminidase-treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate-binding domain of gC-1.