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P G Allen

Publications and source records attributed to P G Allen.

35 records · Page 2Linked to original sources
PubMed1997

Direct quantification of HIV-1 RNA by capillary electrophoresis with laser-induced fluorescence.

HIV-1 RNA was quantitated directly by capillary electrophoresis with laser-induced fluorescence (CE-LIF). CE-LIF was used to analyze cellular RNA and various nucleotide complexes. A fluorescently labeled DNA probe (DNA/RNA complex) in conjunction with thiazole orange intercalator was determined to have optimal stability and sensitivity for RNA analysis. Based on this observation, a hybridization method using a HIV-specific fluorescently labeled probe with analysis by CE-LIF was developed. Plasma samples from a HIV-seropositive patient were lysed to obtain RNA, hybridized with the HIV-specific probe and analyzed by CE-LIF. As little as 19 fg (1710 copies per 1 ml of starting plasma) of HIV RNA can be reliably and quantitatively detected. CE-LIF appears to be an efficient and sensitive method to quantitatively analyze RNA from a variety of sources.

Animals↗
PubMed1997

Functional consequences of disulfide bond formation in gelsolin.

Gelsolin is an actin monomer binding and filament severing protein synthesized in plasma and cytoplasmic forms differing by an N-terminal amino acid extension and a disulfide bond between Cys-188 and Cys-201. To determine whether this bond altered gelsolin regulation or function, oxidized and reduced plasma gelsolins were assayed for severing, monomer binding and nucleation activity at a variety of rate-limiting calcium concentrations. The results indicate that the disulfide bond in domain 2 of gelsolin influences the transmission of information from C-terminal regulatory sites to functional sites in the N-terminus.

Disulfides↗
PubMed1997

Dynamics of filamentous actin organization in the sea urchin egg cortex during early cleavage divisions: implications for the mechanism of cytokinesis.

We have used confocal laser scanning microscopy in conjunction with BODIPY-phallacidin staining of filamentous actin to investigate changes in the quantity and organization of cortical actin during the first two cell cycles following fertilization in eggs of the sea urchin Strongylocentrotus purpuratus. Quantification of fluorescent phallacidin staining reveals that the amount of filamentous actin (F-actin) in the cortex undergoes cyclical increases and decreases during early cleavage divisions, peaking near the beginning of the cell cycle and decreasing to a minimum at cytokinesis. Changes in the content of cortical F-actin are accompanied by the growth and disappearance of rootlet-like bundles of actin filaments which extend from the bases of microvilli that cover the surface of the egg. Actin rootlets reach their maximum degree of development by 20 min postfertilization, and then gradually decrease in number and length over the next 40 min. Small actin rootlets persist until cleavage, disappear during cytokinesis, and reform following division. The formation of actin rootlets requires cytoplasmic alkalization and is inhibited by cytochalasin D. Cytochalasin D washout experiments demonstrate that assembly of the cortical actin cytoskeleton can be blocked until 5 min before the onset of cleavage and still allow normal cytokinesis. These results illustrate the dynamic nature of cortical actin organization during early development and demonstrate that cytokinesis occurs at the point of minimum cortical F-actin content. They further demonstrate that cytokinesis can occur in embryos in which the normal developmental sequence of changes in cortical actin organization has been blocked by treatment with cytochalasin D, suggesting that these changes do not function in the establishment of the contractile apparatus for cytokinesis, but rather serve other developmental functions. Cell Motil. Cytoskeleton 36:30-42, 1997.

Actins↗
PubMed1996

Phalloidin binding and rheological differences among actin isoforms.

Actin is a highly conserved protein in eukaryotes, yet different isoforms of this protein can be found within the same cell. To begin to explore whether isoactin sequence diversity leads to functional differences in actin filaments, we have examined the phalloidin binding kinetics and the bulk rheologic properties of purified actin isoforms from a variety of eukaryotic sources. We observe differences in the phalloidin association kinetics between muscle alpha- and cytoplasmic actins. Phalloidin dissociates from all mammalian actin isoforms tested at the same slow rate, while dissociation from yeast actin is 1 order of magnitude more rapid. The actin isoforms form viscoelastic gels to varying degrees with skeletal muscle alpha-actin gels being the most elastic, smooth muscle alpha- and gamma-actins being less elastic, and beta-actin not forming elastic structures under our experimental conditions. The sequence variation among isoforms is discussed in light of these biophysical and biochemical differences.

Actins↗
PubMed1996

Binding of phosphate, aluminum fluoride, or beryllium fluoride to F-actin inhibits severing by gelsolin.

Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of ATP hydrolysis and the release of the hydrolysis products ADP and inorganic phosphate suggest that phosphate-rich domains exist in newly polymerized filaments. We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluoride and beryllium fluoride, to actin filaments reduces their susceptibility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO4(2-) binding to actin filaments generates this resistant state. We also provide evidence for two different binding sites for beryllium fluoride on actin. Actin has been postulated to contain two Pi binding sites. Our data suggest that they are sequentially occupied following ATP hydrolysis by HPO4(2-) which is subsequently titrated to H2PO4-. We speculate that beryllium fluoride and aluminum fluoride bind to the HPO4(2-) binding site. The cellular consequences of this model of phosphate release are discussed.

Actins↗
PubMed1996

Actin filaments mediate DNA fiber formation in chronic inflammatory airway disease.

Purulent sputum from patients with chronic obstructive pulmonary disease has long been known to contain large DNA-rich fibers believed to impede airway drainage. We present a novel approach to study sputum structure using fluorescence microscopy to confirm the presence of large DNA-rich fibers and visualize for the first time filamentous actin in all sputum samples examined from patients with cystic fibrosis and chronic bronchitis. Both actin and DNA co-localize in the filaments previously identified as DNA alone. Treatment of sputum samples with recombinant human DNase I or the actin-filament-severing protein, gelsolin, both previously found to decrease viscosity, dissolves the sputum fiber bundles. Purified human DNA does not form large fibers alone in vitro but does so in the presence of filamentous actin, and these fiber bundles dissolve when treated with either gelsolin or DNase I. These findings implicate actin-DNA interactions in the pathogenesis of airway disease and identify both polymers as targets for therapy.

Actins↗
PubMed1994

Gelsolin displaces phalloidin from actin filaments. A new fluorescence method shows that both Ca2+ and Mg2+ affect the rate at which gelsolin severs F-actin.

We describe an assay for measuring both the extent and kinetics of the severing of F-actin, based on the enhanced fluorescence emission of tetramethylrhodamine-phalloidin bound to F-actin. The enhanced fluorescence is lost after exposure to active gelsolin by displacement of the phalloidin from actin during severing. This assay requires small amounts of actin and gelsolin, can be used to measure reaction times ranging from 1 to 10(3) s, and does not require covalent modification of either protein. The rate of fluorescence loss is linearly related to the concentrations of both actin and gelsolin. However, the apparent rate constant of the reaction is highly dependent on the divalent cation concentration, varying between 10(4) and 10(6) M-1 s-1 when the [Ca2+] varies between 20 and 200 microM. Addition of Mg2+ increases the apparent rate constant at equivalent Ca2+ concentration. These results suggest that in vitro the rate-limiting step in the severing process is the activation of gelsolin by the binding of Ca2+ and Mg2+ to several low affinity (Kd approximately 100 microM) sites on gelsolin. While activation of gelsolin by Ca2+ is a slow process, the binding and severing of actin occurs at a rate approaching the diffusion limit.

Actins↗
PubMed1994

Reduction in viscosity of cystic fibrosis sputum in vitro by gelsolin.

Obstruction of airways by viscous sputum causes lung damage in patients with cystic fibrosis (CF). Sputum samples from CF patients were shown to contain filamentous actin. Human plasma gelsolin, a protein that severs actin filaments, rapidly decreased the viscosity of CF sputum samples in vitro. Gc globulin and deoxyribonuclease I, proteins that sequester monomeric actin but do not sever actin filaments, were less efficient than gelsolin in diminishing sputum viscosity. These results suggest that gelsolin may have therapeutic potential as a mucolytic agent in CF patients.

Actins↗
PubMed1994

Remodeling of neutrophil phospholipids with 15(S)-hydroxyeicosatetraenoic acid inhibits leukotriene B4-induced neutrophil migration across endothelium.

5-Lipoxygenase products, such as leukotrienes, are important stimuli for leukocyte-mediated tissue injury in acute inflammation. 15-Hydroxyeicosatetraenoic acid (15-HETE) is an eicosanoid generated by a variety of cell types via the actions of 15-lipoxygenases and, in addition, cyclooxygenases and epoxygenases. 15-HETE levels are frequently elevated at sites of inflammation, and extracellular 15(S)-HETE is esterified rapidly into neutrophil (PMN) phospholipids in vitro to levels that are comparable with arachidonic acid. We present evidence that remodeling of PMN phospholipids with 15(S)-HETE stereoselectively inhibits PMN migration across endothelium in response to leukotriene B4 (LTB4) and other chemoattractants. Esterified 15(S)-HETE causes a striking reduction in the affinity of LTB4 cell-surface receptors for their ligand and inhibition of LTB4-triggered stimulus-response coupling. As a result of these actions, esterified 15(S)-HETE attenuates the cytoskeletal rearrangements and CD11/CD18-mediated adhesive events that subserve directed locomotion of PMN across endothelium. These observations indicate that products of the 5-lipoxygenase and 15-lipoxygenase pathways can exert counterbalancing influences on PMN trafficking across endothelium. They suggest that 15(S)-HETE may be a potent endogenous inhibitor of PMN-endothelial interactions in vivo and serve to limit or reverse acute inflammation.

Antigens, CD↗
PubMed1993

Selective binding of gelsolin to actin monomers containing ADP.

The rate of reaction and the stoichiometry of binding between gelsolin and actin monomers depends on adenine nucleotides. In the presence of Ca2+ but not Mg2+, gelsolin retains the ability to sever actin filaments when incubated for more than 20 min with an excess of G-actin in the presence of ATP but loses severing activity within seconds when mixed with G-actin in ADP. Immunoprecipitation of gelsolin removes more actin from ADP than from ATP solutions. Monomeric ATP-actin in 2 mM MgCl2 and 150 mM KCl slowly destroys the filament-severing activity of gelsolin with kinetics that are first order in actin concentration and with an apparent bimolecular rate constant of 0.021 +/- 0.007 microM-1 s-1. Coincident with the slow complex formation in MgCl2, the actin bound to the calcium-sensitive actin binding domain of gelsolin hydrolyzes its ATP to ADP. These results suggest a further level of gelsolin regulation and a functional similarity between actin and GTP-binding proteins.

Actins↗
PubMed1993

Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement.

The activation of gelsolin by calcium has been postulated to be involved in the receptor-mediated reorganization of the actin cytoskeleton, but cytoskeletal reorganization can also occur in cells with intracellular Ca2+ clamped at nanomolar levels. Fluorescence measurements using Fura-2 show that at pH 7.4, the Ca2+ requirement for gelsolin activation in vitro is higher than previously reported, with half-maximal activation of severing and nucleation occurring at 10 microM Ca2+. The Ca2+ requirement for gelsolin activity decreases at more acid pH and is approximately 3 microM at pH 6.5. At pH below 6.0, gelsolin no longer requires Ca2+ for activity and severs actin filaments, binds two actin monomers, and nucleates filament formation in EGTA-containing solutions. The pH-activated severing activity is inhibited by mixed lipid vesicles containing phosphatidylinositol 4,5-bisphosphate. A Ca(2+)-sensitive fragment consisting of the first 135 amino acids of human cytoplasmic gelsolin also demonstrates severing activity at pH < 6.0 in the absence of Ca2+. In contrast, the gelsolin homologs severin and villin maintain Ca2+ regulation of severing activity at low pH. These differences suggest that activation of gelsolin at low pH cannot be explained merely by destabilization of F-actin. The difference in diffusion constants of gelsolin measured at pH 5.5 and 6.5, as determined by dynamic light scattering, suggests that the molecule undergoes a shape change similar to that reported upon binding Ca2+ at neutral pH. These results suggest a mechanism by which gelsolin may be activated in vivo under conditions where Ca2+ transients do not occur.

Actins↗
PubMed1992

Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin.

The polyphosphoinositides phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) inactivate the actin filament-severing proteins villin and gelsolin and dissociate them from monomeric and polymeric actin. A potential polyphosphoinositide- (PPI) binding site of human plasma gelsolin regulating filament severing has been localized to the region between residues 150-169 and to the corresponding region in villin which occurs in the second of six homologous domains present in both proteins. Synthetic peptides based on these sequences bind tightly to both PIP and PIP2, in either micelles or bilayer vesicles, compete with gelsolin for binding to PPIs, and dissociate gelsolin-PIP2 complexes, restoring severing activity to the protein. These peptides also bind with moderate affinity to F-actin, suggesting that inactivation of the severing function of the intact proteins by PPIs results from competition between actin and PPIs for a critical binding site on gelsolin-villin. The PPI-binding peptides contain numerous basic amino acids, but their effects on PPIs are far greater than those of Arg or Lys oligomers, a highly basic peptide derived from the calmodulin-binding site of myristoylated, alanine-rich kinase C substrate protein, or the 5-kDa actin-binding protein thymosin beta-4, suggesting that specific aspects of the primary and secondary structure of these basic peptides are important for their interaction with the acidic headgroups of PPIs. In addition to elucidating the structure of PIP2-binding sites in gelsolin, the results describe a sensitive assay for phosphoinositide-binding molecules based on their ability to prevent inhibition of gelsolin function.

Amino Acid Sequence↗
PubMed1992

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.

Animals↗
PubMed1990

Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast.

We have examined the initial events in phagocytosis by Acanthamoeba castellanii in order to understand this process at the molecular level and have determined that phagocytosis in this organism is mediated by a receptor which recognizes mannose-rich elements in the particle to be phagocytosed. We demonstrate that the binding and internalization of yeast particles can be inhibited by the sugars (D(+)-mannose and D(-)-fructose in a stereospecific, concentration-dependent manner. This inhibition is specific; these sugars did not inhibit the uptake of latex beads by this organism. Using mannosylated neoglycoproteins, which are much more potent inhibitors of particle binding as compared with the free sugar, we demonstrate the presence of a receptor on the amoeba cell surface which is necessary for the binding of yeast as the initial event of phagocytosis. The Acanthamoeba mannose receptor also appears to be able to mediate the delivery of soluble mannose-rich molecules to a degradative compartment such as the lysosome. Knowledge of this receptor will allow a better understanding of the molecular events of phagocytosis.

Acanthamoeba↗
PubMed1990

Phagocytosis in Acanthamoeba: II. Soluble and insoluble mannose-rich ligands stimulate phosphoinositide metabolism.

The generation of second messengers during phagocytosis of yeast by Acanthamoeba castellanii was examined. The kinetics of binding and internalization of yeast by Acanthamoeba were measured and this was compared with the generation of known second messengers. We observed stimulated degradation of PI-4, 5-P2 to 1,4,5 IP3 with kinetics similar to that observed for the binding of yeast to amoeba. Similar production of IP3 could be induced upon treatment with a soluble mannosylated glycoprotein. We propose that the Acanthamoeba mannose receptor stimulates the degradation of PI-4, 5-P2 to 1,4,5 IP3 as an initial event in phagocytosis.

Acanthamoeba↗
PubMed1981

Sensory specificity of apparent motion.

Previous reports of intermodal apparent motion claim that the various senses contribute to or are under the guidance of a single synthesizing or organizing system, a common sense, and that under proper circumstances the separate heteromodal stimuli may be combined into a novel perceptual object. Our first experiment suggests that the term intermodal apparent motion has been misapplied in the past to one of the many idiosyncratic interpretations to which relatively meaningless patterns of heteromodal stimulation lend themselves. In our second experiment, we found that a properly timed visual stimulus can facilitate the perception of auditory apparent motion without its peculiarly auditory qualities of the moving percept. We find no evidence for a common or suprasensory organizing principle that integrates the separate visual and auditory stimuli.

Auditory Perception↗