Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.     

The influence of phenelzine and tranylcypromine on the release of prostaglandins from the rat mesenteric vascular bed

We investigated the effects of phenelzine and tranylcypromine on the release of prostacyclin, thromboxane A2, prostaglandin E2, and prostaglandin E1 from the isolated perfused rat mesenteric vascular bed. Perfusion of the preparation with phenelzine in concentrations of 15, 45, and 135 microM for 150 min led to attenuated release of all four prostaglandins measured. Inhibition generally occurred with the lowest dose used and was most prominent with the highest concentration. Tranylcypromine also decreased prostaglandin formation. However, low doses were not effective in the suppression of prostacyclin release. Both drugs had an inhibitory effect on production of prostaglandin E1, which is a metabolite of dihomo-gamma-linolenic acid, the precursor of arachidonic acid, but this was only shown to be significant with phenelzine. In this work we demonstrate that phenelzine and tranylcypromine have an inhibitory effect on the production of 2-series prostaglandins derived from arachidonic acid, and possibly a similar effect on prostaglandins of the 1-series derived from dihomo-gamma-linolenic acid.     

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.     

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.     

Pertussis toxin as a probe of neutrophil activation

In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed.     

Alpha-L-fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property

Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.     

Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurites show pathway specificity but lack directional specificity or predetermined lengths in Xenopus embryos

The earliest outgrowth of nerve fibers from identified spinal neurons labeled with horseradish peroxidase (HRP) was traced along surgically rearranged pathways in the central nervous system (CNS) of Xenopus embryos. Parts of the CNS were misaligned or inverted rostrocaudally by grafting a segment of labeled spinal cord in place of the same or different spinal cord segment of an unlabeled embryo or by joining two rostral half embryos (head-to-head) or two caudal half embryos (tail-to-tail), one half of which was derived from a labeled embryo in each combination. Donor embryos were labeled by injection of HRP into a selected blastomere at the 16- or 32-cell stage. Host embryos were unlabeled. Grafts from labeled donors to unlabeled host embryos were made at early neural tube stages before outgrowth of any nerve fibers had started (Jacobson and Huang, 1985). Routes taken by labeled nerve fibers growing into unlabeled CNS were observed at later stages, and the rates of nerve fiber elongation were calculated. Labeled nerve fibers were normal in appearance, and elongated without branching, at normal rates (22-71 micron/h). In head-to-head and tail-to-tail embryos and in embryos with inverted spinal cord grafts, nerve fibers continued elongating without branching in the direction opposite to normal in the CNS. Many fibers reached lengths that were far greater than normal. No reorientation of such maldirected nerve fibers was seen. These results indicate that nerve fiber elongation is not guided by axially polarized pathway cues or markers and that nerve fibers do not grow to predetermined lengths. However, neurites preferred to grow along stereotyped nerve fiber pathways even when forced to grow in the wrong direction or when confronted with nonneural tissue.     

Further characterization of the vesicular stomatitis virus temperature-sensitive O45 mutant: intracellular conversion of the glycoprotein to a soluble form.

Reexamination of the viral products of tsO45, a glycoprotein mutant of vesicular stomatitis virus, showed that at 39 degrees C there was a conversion of the glycoprotein (G) to a truncated, soluble form, Gs, which subsequently appeared in the extracellular medium. The half-life for this intracellular conversion and extracellular appearance was about 2 h at 39 degrees C. Gs was precipitated by a monoclonal antibody to the ektodomain but not by an antipeptide serum made against the first 15 amino acids at the carboxy terminus of G. Gs was also resistant to endoglycosidase H digestion. On the basis of pulse-chase experiments, the generation of Gs most probably occurred in the rough endoplasmic reticulum. This additional phenotype of the tsO45 mutant provides another approach for studying the generation and subsequent transport of a secreted protein in fibroblast cells.