Cyclo-oxygenase blockers influence the effects of 15-lipoxygenase metabolites of arachidonic acid in isolated canine blood vessels

In canine saphenous veins both the 15-hydroxy- and 15-hydroperoxy derivatives of arachidonic acid, 15HETE and 15HPETE, caused endothelium-independent contractions which were not affected by a variety of classical receptor antagonists. These contractions were markedly augmented by cyclooxygenase blockers; nifedipine, which did not influence the contractions induced by lipoxygenase products, inhibited the potentiating effect of indomethacin. In the veins, 15HETE and 15HPETE also induced spotaneous rhytmic contractions which persisted after several washings but could be blocked by inhibitors of clyclooxygenase. In coronary, splenic, renal and femoral arteries, 15HETE and 15HPETE caused contractions which were also augmented by indomethacin and were dependent on the influx of extracellular calcium as they were inhibited by verapamil. Both 15-lipoxygenase metabolites evoked relaxations during contractions induced by prostaglandin F_2α or the thromboxane-mimetic U46619. These relaxations were not endothelium-dependent but were inhibited by indomethacin; they did not occur when the initial contractions were caused by K⁺, norepinephrine or 5-HT. Our results illustrate multiple vascular actions of 15HETE and 15HPETE in dog blood vessels.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-β- -galactopyranoside and methyl O-(3-deoxy-3-fluoro-β- -galactopyranosyl)-(1→6)-O-β- -galactopyranosyl-(1→6)-β- -galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α- -galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β- -galactopyranoside (4) gave a fully acetylated (1→6)-β- -galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α- -galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β- -galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β- -galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β- -galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β- -galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β- -galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Binding and internalization of heparin by vascular smooth muscle cells

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.     

Accumulation and Subcellular Localization of alpha-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons

We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) α-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with α-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that α-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.     

Plasmid-dependent inhibition of growth of bacteriophage T4 ndd mutants.

Mutants of bacteriophage T4 that fail to induce nuclear disruption (ndd mutants) are unable to grow in the wild-type Escherichia coli strain CT447. This inhibition of the growth of ndd mutants occurs only in the presence of a large (ca. 80-megadalton) plasmid resident in CT447 cells.     

DNA analysis and sorting of rat testis cells using two-parameter flow cytometry

By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

Introduced reindeer and their effects on the vegetation and the epigeic invertebrate fauna of South Georgia (subantarctic)

Summary The introduced reindeer of South Georgia have had a serious impact on the vegetation throughout the range of three populations on the island. Exclosure experiments in areas where the reindeer were introduced have resulted in a dramatic change in the composition of the protected vegetation. Poa flabellata (the major winter food) and Acaena magellanica (a major summer food) have recovered to their former status inside the exclosures, while Deschampsia antarctia and the introduced grass Poa annua tolerate grazing and trampling and have spread over the grazed areas. Festuca contracta and Rostkovia magellanica are not eaten by the reindeer. Trampling has resulted in a high proportion of bare soil and peat in sites freely accessible to reindeer. However, the changes in the vegetation have not had such a significant effect on the associated invertebrate fauna. Thus in grazed and protected areas the faunistic composition is qualitatively similar, although there are quantitative differences, and some of the trends can probably be attributed to the presence of the reindeer. Compared with reindeer-free areas, the abundance of the perimylopid beetle Hydromedion sparsutum (a major primary decomposer) is reduced. The frequency of their egg parasite Notomymar aptenosoma (Hymenoptera, Mymaridae) increases. Consequently the ratio of perimylopoids to mymarids found in pitfall traps shifts from 1:0.01 (ungrazed areas) to 1:0.54 (grazed areas). Also the frequency of sciarids was found to be higher in reindeer areas. The larvae of these probably introduced gnats do not play a role in the natural terrestrial ecosystem of South Georgia, but in reindeer areas they appear to establish larger populations because they are able to live deeper in the soil and in hardened substrates. There is also a shift in the ratio between Collembola (major prey) to spiders from 1:1.3 (ungrazed areas) to 1:0.82 (grazed areas), for animals collected in pitfall traps. This may be a result of the trampling effect of the reindeer.