Production of inflammatory mediators by human macrophages obtained from ascites

Ascites is a readily available source of human macrophages (M phi), which can be used to study M phi functions in vitro. We characterized the mediators of inflammation produced by human peritoneal M phi (hp-M phi) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was found to be lipopolysaccharide (LPS) concentration dependent (0-10 micrograms/ml) with a maximal production at 10 micrograms/ml and also dependent on the time of exposure to the stimulus (0-36 h). IL-1 beta, IL-6 and TNF-alpha production after LPS administration reached a plateau at 24 h. In vitro stimulation for 24 h with LPS does not influence the eicosanoid production from endogenous arachidonate. 13 min of exposure of the cells to the calcium ionophore A23187 gives a significant increase in eicosanoid production from both exogenous and endogenous arachidonate. The main eicosanoids produced are the 5-lipoxgenase products LTB4 and 5-hydroxyeicosatetraenoic acid (HETE). The increase in production of the other eicosanoids is not significant. The eicosanoid production depends on the stimulus concentration. The optimal A23187 concentration is 1 microM. Oxygen radical production was measured in the M phi by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded hp-M phi increases significantly after 15 min. We conclude that LPS stimulation of hp-M phi from liver disease results in similar production of IL-1 beta, IL-6 and TNF-alpha, but that the profile of the eicosanoid production of these M phi stimulated with LPS and A23187 differs from M phi of other origin and species.     

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.     

Cardiorespiratory effects of induction and maintenance of anesthesia with ketamine-midazolam combination, with and without prior administration of butorphanol or oxymorphone

Cardiorespiratory effects of an IV administered bolus of ketamine (7.5 mg/kg of body weight) and midazolam (0.375 mg/kg) followed by IV infusion of ketamine (200 micrograms/kg/min) and midazolam (10 micrograms/kg/min) for 60 minutes was determined in 6 dogs. Ketamine-midazolam combination was administered to dogs on 3 occasions to determine effects of prior administration of IV administered saline solution (1 ml), butorphanol (0.2 mg/kg), or oxymorphone (0.1 mg/kg). The infusion rate of ketamine and midazolam was decreased by 25% for anesthetic maintenance after opioid administration. There were no significant differences in cardiorespiratory variables after saline solution or butorphanol administration; however, oxymorphone caused significant (P < 0.05) increases in mean arterial blood pressure, systemic vascular resistance, and breathing rate. Bolus administration of ketamine-midazolam combination after saline solution caused significant (P < 0.05) increases in heart rate, mean arterial blood pressure, cardiac index, mean pulmonary blood pressure, venous admixture, and significant decreases in stroke index, pulmonary capillary wedge pressure, arterial and mixed venous oxygen tension, arterial oxygen content, and alveolar-arterial oxygen gradient. Opioid administration was associated with significantly (P < 0.05) lower values than was saline administration for heart rate, mean arterial blood pressure, and arterial and mixed venous pH and with higher values for stroke index, pulmonary capillary wedge pressure, and arterial and mixed venous carbon dioxide tension. Prior oxymorphone administration resulted in the highest (P < 0.05) values for mean pulmonary blood pressure, venous admixture, and arterial and mixed venous carbon dioxide tension, and the lowest values for arterial oxygen tension, and arterial and mixed venous pH. Each treatment provided otherwise uncomplicated anesthetic induction, maintenance, and recovery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platinosomes produced in cultured cells by platinum coordination complexes

Cells in culture were exposed to cis-dichlorodiammineplatinum (II) and platinum-uracil blue. No platinum could be demonstrated in an intracellular location morphologically or by electron-probe x-ray analysis in the case of the former compound. However, platinum was readily demonstrated in phagosomes or phagolysosomes after exposure of cells to platinum-uracil blue.     

Shared reaction in solid-phase immunoassay for estriol determination

In the search of factors responsible for the experimental difficulties in developing accurate and sensitive solid-phase immunoassay of steroids, an experimental model has been set up for the study of nonspecific interaction of the steroid analyte with the coating protein. Along with the development of a highly sensitive enzyme-linked, solid-phase immunoassay for estriol measurement, we observed evidence of shared reactions. This property, to our knowledge not previously described for monomeric, low-molecular-weight antigens like estrogens, has been attributed to the presence of bovine serum albumin, which is capable of binding estrogens through hydrophobic interactions. The addition of estriol in solution in large excess did not reach a complete inhibition of the binding, so the possibility was excluded that the antibody simply binds to the adsorbed estrogen. The simplest explanation for the occurrence of the reaction is the hypothesis that a family of antigen determinants arises when the estriol is conjugated to a protein carrier. The corresponding antibodies are revealed only when the estrogen participates to the actual analytical system in the form of a steroid-protein conjugate. In the experiment, the estriol has been recognized as being coupled with one or more amino acid side chains present around its site of covalent linkage to the immunogen protein. The discussed results may be of help in developing a solid-phase immunoassay of small antigens as steroids, but also in applying the hybridoma and phage display technologies, the screening methods of which are based on sensitized solid phases.     

Stress and strain in the flight muscles as constraints on the evolution of flying animals

The authors describes a new device for external tissue extension (ETE) which will be able to replace or complement tissue expanders. The device consists of many ETE units, each unit consisting of a needle and two friction stoppers counted on a silicone string. Application, optimal tension and final surgical procedure are described. The indications are the same as for tissue expanders, e.g. scars, naevi and previous skin grafts, and also concern the closure of acute fasciotomies. The advantages are numerous: very simple technique, application under local anaesthetics, faster cutaneous profits (5-6 days), inexpensive total treatment, low complication rate.     

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.     

Enantiomers of myo-inositol-1,3,4-trisphosphate and myo-inositol-1,4,6 -trisphosphate: stereospecific recognition by cerebellar and platelet myo-inositol-1,4,5-trisphosphate receptors

The naturally occurring tetrakisphosphate myo-inositol-1,3,4, 6-tetrakisphosphate [Ins(1,3,4,6)P4] was able to release Ca2+ from the intracellular stores of permeabilized rabbit platelets but was 40-fold less potent than D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ca2+ releasing activity of Ins(1,3,4,6)P4 was rationalized by envisaging two alternative receptor binding orientations in which the vicinal D-1,6-bisphosphate of Ins(1,3,4,6)P4 mimics the D-4,5-bisphosphate in the Ins(1,4,5)P3 binding conformation. This rationalization predicted that Ins(1,4,5)P3 regioisomers [i.e, D-myo-inositol -1,4,6-trisphosphate [D-Ins(1,4,6)P3] and D-myo-inositol-1,3,6 -trisphosphate [D-Ins(1,3,6)P3]] should also possess Ca(2+)-releasing activity. The unambiguous total synthesis of the enatiomers of Ins(1,4,6)P3 [i.e., D-Ins(1,4,6)P3 and D-Ins(3,4,6)P3] and the enatiomers of Ins(1,3,4)P3 [i.e., D-Ins(1,3,6)P3 and D-Ins(1,3,4)P3] allowed an examination of this prediction. D-Ins(1,4,6)P3 released Ca2+ from the intracellular stores of permeabilized platelets and was only 2-3-fold less potent than Ins(1,4,5)P3. D-Ins(1,3,6)P3 [alternative nomenclature, L-Ins(1,3,4)P3] also released Ca2+ but was 12-fold less potent than Ins(1,4,5)P3. Both D-Ins(1,4,6)P3 and D-Ins(1,3,6)P3 displaced specifically bound [3H]Ins(1,4,5)P3 from the Ins(1,4,5)P3 receptor on rat cerebellar membranes. In contrast, however, D-Ins(3,4,6)P3 [alternative nomenclature, L-Ins(1,4,6)P3] and D-Ins(1,3,4)P3 neither possessed Ca(2+)-releasing activity nor displaced [3H]Ins(1,4,5)P3. The ability of D-Ins(1,3,6)P3 to release Ca2+ in permeabilized platelets is in contrast to its apparent lack of Ca(2+)-mobilizing activity previously reported in rat basophilic leukemic cells. The possibility that this is a reflection of the different Ins(1,4,5)P3 receptor subtypes possessed by these two cell types is discussed.     

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme HindIII, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori, and their relative discriminatory abilities were assessed. Four probes, (GACA)4, (GT)8, (GTG)5 and (GGAT)4, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)4-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori.