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At least 415 records · Page 23Linked to original sources
PubMed1992

Triazolam blocks the initial rotational effects of quinpirole but permits the later developing reduction of dopamine D2-mediated rotational behavior and dopamine D2 receptors.

Continuous infusion of the dopamine D2 receptor agonist quinpirole into mice with unilateral striatal 6-hydroxydopamine lesions initially produces a supersensitive rotational behavior. This is followed by reductions of dopamine D2-mediated behavior and dopamine D2 receptors. In this study we attempted to determine if it is possible to inhibit the acute increase in D2-mediated behavior while still allowing the reduction of D2-mediated behavioral responses and dopamine D2 receptors to occur. Mice were implanted with Alzet minipumps containing either quinpirole alone or quinpirole combined with the GABA receptor modulator triazolam or the dopamine D2 receptor antagonist sulpiride, and rotational behavior was monitored for the 6 days of infusion. The pumps were then removed, and D2 receptors in striatal membranes were determined. Triazolam completely blocked the initial rotational behavior normally induced by implanting quinpirole. However, the quinpirole-induced reduction of D2-mediated behavioral responses and D2 receptors still occurred. Continuous infusion of sulpiride also inhibited the rotational behavior produced by quinpirole, but it prevented the reduction of dopamine D2 receptors. We conclude that up-regulated dopamine receptors and dopaminergic behaviors can be reversed by the continuous administration of a dopamine receptor agonist and that this reversal can occur without producing an initial exacerbation of dopaminergic responses. These results suggest that this type of treatment regimen might be useful for treating clinical conditions associated with dopaminergic supersensitivity.

Analysis of Variance↗
PubMed1993

Antibodies to fluorylsulfonylbenzoyladenosine permit identification of protein kinases.

Antibodies were raised to fluorylsulfonylbenzoyladenosine (FSBA) coupled to carrier proteins. Affinity purified sera are shown to specifically detect protein kinase C-beta 1 treated with FSBA. Immunodetection is specific to modification at the ATP binding site as judged by the ability of ATP to block subsequent detection. The antisera are shown to react with a number of U937 cell proteins fractionated on Mono Q and treated with FSBA; the majority of these immunoreactions are prevented by inclusion of ATP in the FSBA treatments. One of these is positively identified as PKC-beta 1.

Adenosine↗
PubMed1994

A mutation blocking the formation of membrane or periplasmic endogenous and exogenous c-type cytochromes in Escherichia coli permits the cytoplasmic formation of Hydrogenobacter thermophilus holo cytochrome c552.

A mutant of Escherichia coli, JCB606, shown to be pleiotropically deficient in the formation of endogenous membrane and periplasmic c-type cytochromes, synthesised the apo form of the exogenous cytochrome c550 from Paracoccus denitrificans, but not the holo form. In contrast, a cytoplasmically located holo form of Hydrogenobacter thermophilus cytochrome c552 was found in E. coli JCB606. These findings support the proposition that the formation of the cytoplasmic H. thermophilus cytochrome c552 in E. coli does not involve the physiological pathway of c-type cytochrome biosynthesis in E. coli and that the haem insertion may be catalysed.

Cytochrome c Group↗
PubMed1976

Disc-crossed immunoelectrophoresis. A simple "laying-on" technique permitting the use of commercially available agarose.

A technique, disc-crossed immunoelectrophoresis, is described to provide a simple and convenient means of performing electrophoresis in polyacrylamide gel in a discontinuous buffer system (disc electrophoresis) followed by electrophoresis into antibody-containing agarose gel. A simple washing of the polyacrylamide gel in distilled water combined with a "laying-on" method, seems to be a satisfactory and rapid way to obtain immunoprecipitation patterns of high quality using commercially available agarose in the second electrophoresis.

Electrophoresis, Agar Gel↗
PubMed1984

Cultivation of murine bone marrow macrophages in sponges: a method that permits recovery of viable cultured cells.

Various investigators have cultured murine bone marrow or peritoneal cells in vitro on glass or plastic surfaces with the ultimate aim of retrieving adherent macrophages for morphologic and functional evaluation. The removal of these adherent macrophages by conventional techniques has been consistently accompanied by low yield and significant cell damage. We report here a simple technique for culturing murine bone marrow cells in gelatin sponges (Spongostan and Gelfoam) in growth medium containing 10% fetal bovine serum and 10% L-cell conditioned medium. Viable cells were retrieved from the sponges in 10 min by digestion with collagenase. The in situ growth kinetics were similar to those found for cells cultured on plastic dishes. The recovered cells were adherent, phagocytic, positive for Fc gamma receptors, and had esterase activity.

Animals↗
PubMed1984

A freezing method for cell fusions to distribute and reduce labor and permit more thorough early evaluation of hybridomas.

A method is described whereby cell fusions can be bulk-frozen shortly after the hybridization step. Recoveries are shown to be comparable to those obtained for control hybridomas cultured without freezing. Advantages are discussed in terms of labor distribution and antibody assay and evaluation strategies. It is further shown that peritoneal feeder cell preparations can be conveniently frozen as a means of workload reduction.

Animals↗
PubMed1987

Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens.

Fixation and immunofluorescent staining methods were developed for analyzing intracellular antigens with the cell flow cytometer. Fixing cell suspensions with 100% methanol provided best preservation of morphology, lowest fluorescent background staining and most intense specific immunofluorescence. Immunoglobulins present in B cell lines that were representative of different developmental stages could be distinguished quantitatively. Fluorescence histograms were compared with fluorescence microscope presentation of stained cells. Intracellular antigens that reacted with monoclonal antibodies could also be evaluated by flow cytometry. This method was utilized to assess plasmacyte development in mouse spleen cell cultures after stimulation with lipopolysaccharide.

Animals↗
PubMed1989

Treatment of human muscle creatine kinase with glutaraldehyde preferentially increases the immunogenicity of the native conformation and permits production of high-affinity monoclonal antibodies which recognize two distinct surface epitopes.

Treatment of human muscle creatine kinase (MM-CK) with glutaraldehyde produced highly aggregated forms which retained the native antigenic structure. Immunization of BALB/c mice with CK aggregates instead of untreated CK produced over ten-fold higher titres of antibody against native CK without increasing the titres of antibody against denatured enzyme. Production of high-affinity monoclonal antibodies specific for both the muscle isoenzyme and the native conformation became possible where the use of untreated CK had failed. Four monoclonal antibodies have been characterized by an epitope mapping technique and compared with a commercially available monoclonal antibody. One antibody has a much higher affinity for MM-CK than the other three and the commercial antibody. Competition studies show that it also recognizes a different epitope on the CK surface from the other three monoclonal antibodies which bind to the same surface region as the commercial antibody. Immunoassays based on the high affinity antibody can easily measure less than 1 ng of CK, a sensitivity comparable to, or better than, standard enzymatic assays.

Antibodies, Monoclonal↗
PubMed1995

Novel isolation and purification method permitting functional cytotoxicity studies of macrophages from milky spots in the greater omentum.

Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages are the predominant cell type forming a distinct population of cells. To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro. In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in collagenase/DNase suspension and filtered. Subsequently, macrophages were isolated and purified using flow cytometry by sorting unstained cells on the basis of size and internal complexity. Macrophages and other cells were identified by routine May-Grünwald-Giemsa staining and by immunophenotyping with the specific macrophage monoclonal antibody ED 1. Furthermore, macrophage subtypes were characterized by ultrastructural analysis. Functional cytotoxicity of the isolated macrophages was assayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay. From three greater omenta of healthy rats 1.16 +/- 0.16 x 10(6) macrophages were isolated with a purity of 83 +/- 2% and a viability of > or = 96%. The macrophages were of the exudate (monocytic), exudate-resident and resident cell type and were in equal proportions. The contaminating cells were mainly mesothelial. A maximum cytotoxicity of approximately 30% was reached with the macrophage fraction at an effector-to-target ratio of 10. Furthermore, it was established that the mesothelial cells did not exhibit cytotoxicity.

Animals↗
PubMed1995

The use of dedicated peptide libraries permits the discovery of high affinity binding peptides.

The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been obtained from a phage display peptide library (Schellekens et al., 1994). Binding studies with different length variants of this peptide identified a 12mer as a suitable lead compound for our library study. Two incomplete dedicated resin-bound synthetic peptide libraries were generated. Both consisted of 2 x 10(6) 12mers, in which positions were alternately fixed (amino acids identical to the lead sequence) and random. The libraries were screened with mAb A16 and beads with binding peptides were sequenced using Edman degradation. This resulted in a unique peptide binding motif, essentially comprising a 7mer core sequence. Comparison of the sequence of the natural epitope with the binding motif revealed that its sequence was identical to the motif except for one position. Substitution of a methionine in the natural epitope by a tyrosine or a phenylalanine at that position, as dictated by the motif, resulted in a peptide with an affinity for binding to mAb A16 about 50 times higher than that of the natural epitope. Thus, if a lead sequence is available, the use of incomplete, dedicated synthetic peptide libraries provides a fast and powerful tool for the detection of high affinity peptides.

Amino Acid Sequence↗
PubMed1988

Barnase and barstar. Expression of its cloned inhibitor permits expression of a cloned ribonuclease.

Barnase is the extracellular ribonuclease of Bacillus amyloliquefaciens and barstar its specific intracellular inhibitor. The gene for barstar has now been cloned and sequenced. When the wild-type gene for barnase is reconstructed from its previously cloned parts on the same plasmid as the barstar gene, the lethal effect of its expression is suppressed. A plasmid has been devised which directs the secretion of 100 mg per active barnase liter by Escherichia coli and another which provides large (500 to 1000 mg/l) yields of barstar. The structure of these plasmids and the derived 89 amino acid sequence of barstar are reported.

Amino Acid Sequence↗