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J M Robinson

Publications and source records attributed to J M Robinson.

At least 55 records · Page 3Linked to original sources
PubMed1998

Ultrasmall immunogold particles: important probes for immunocytochemistry.

In this article, we review the immunocytochemical literature with respect to a comparison between conventional colloidal gold and ultrasmall gold particles as immunoprobes. We discuss the relative advantages and disadvantages of each of these types of particles for immunocytochemical applications. We present results from our own laboratories, in which we compared these immunoprobes in selected experimental situations. In addition, we discuss our work on the use of a fluorescently labeled ultrasmall immunoprobe for correlative microscopy.

Fluorescent Dyes↗
PubMed1998

Functional separation of pseudopod extension and particle internalization during Fc gamma receptor-mediated phagocytosis.

Receptors for the Fc portion of immunoglobulin (Ig)G (Fc gamma R) mediate phagocytosis of IgG-opsonized particles by a process that can be divided into four major steps: receptor-ligand binding, pseudopod extension, internalization, and lysosomal fusion. We have expressed single classes of Fc gamma R in COS fibroblasts to examine the structural determinants necessary to complete the four steps of phagocytosis. Using phase contrast, fluorescence, confocal, and electron microscopy we have demonstrated that Fc gamma R-expressing COS cells can phagocytose in a manner similar to that of professional phagocytes. We have further analyzed the capacity of the three classes of Fc gamma R to phagocytose, placing special emphasis on the Fc gamma RIA-gamma chain complex, which allowed us to examine independently the roles of the ligand-binding unit (Fc gamma RIA) and the signaling unit (gamma chain). We found that receptor complexes containing a conserved tyrosine activation motif (ITAM), as found in the cytoplasmic domain of Fc gamma RIIA and in the gamma chain associated with Fc gamma RIA and Fc gamma RIIIA, readily internalized target particles. In contrast, Fc gamma RIA alone, having no ITAM, was unable to internalize target particles efficiently, but did mediate pseudopod extension. Cotransfection of gamma chain with Fc gamma RIA restored the ability of the receptor to internalize target particles. A mutant Fc gamma RIA in which the cytoplasmic domain had been deleted was also capable of mediating pseudopod extension, showing that neither the gamma chain nor the cytoplasmic domain of Fc gamma RIA were required for this step. Cytochalasin D, an inhibitor of actin polymerization, blocked particle internalization by all Fc gamma R, but did not block pseudopod extension. Staining the Fc gamma RIA COS cells for F-actin and for tyrosine phosphoproteins, we found that actin did not polymerize during Fc gamma RIA-mediated pseudopod extension, nor were tyrosine kinases activated. Our data suggest that pseudopod extension and internalization are functionally distinct steps mediated through different pathways.

Actins↗
PubMed1998

Arachidonic acid activates NADPH oxidase by a direct, calmodulin-regulated mechanism.

In intact neutrophils, arachidonic acid rapidly and transiently activated NADPH oxidase-mediated superoxide generation. Inhibitors of protein kinases (staurosporine and H-7) failed to markedly attenuate arachidonic acid-stimulated superoxide generation. Conversely, the calmodulin antagonist W-7 blocked this arachidonic acid-stimulated response. Similarly, diphenylene iodonium potently inhibited superoxide release. These results suggest that arachidonic acid directly activates the membrane-associated NADPH oxidase and calmodulin and/or calmodulin-binding proteins are required for the assembly of the active oxidase.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Valaciclovir for the suppression of recurrent genital herpes simplex virus infection: a large-scale dose range-finding study. International Valaciclovir HSV Study Group.

A randomized, double-blind study of valaciclovir for suppression of recurrent genital herpes was conducted among 1479 immunocompetent patients. Patients were randomized to receive valaciclovir (250 mg, 500 mg, or 1 g once daily, or 250 mg twice daily), acyclovir (400 mg twice daily), or placebo, for 1 year. All valaciclovir dosages were significantly more effective than placebo at preventing or delaying recurrences (P < .0001). There was a dose-response relationship (P < .0001) across the once-daily valaciclovir regimens. Twice-daily valaciclovir and acyclovir were similar in effectiveness. Subgroup analysis showed that patients with a history of < 10 recurrences per year were effectively managed with 500 mg of valaciclovir once daily. One gram of valaciclovir once daily, 250 mg of valaciclovir twice daily, or 400 mg of acyclovir twice daily were more effective in patients with > or = 10 recurrences per year. Safety profiles of all treatments were comparable. Thus, valaciclovir is highly effective and well tolerated for suppression of recurrent genital herpes. Once-daily regimens offer a useful option for patients who require suppressive therapy for management of genital herpes.

Acyclovir↗
PubMed1998

Freeze-fracture cytochemistry: a new method combining immunocytochemistry and enzyme cytochemistry on replicas.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.

Adult↗
PubMed1998

Correlative microscopy using FluoroNanogold on ultrathin cryosections. Proof of principle.

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for high-resolution correlation between immunofluorescence and electron microscopy.

Frozen Sections↗
PubMed1998

Identification of intracellular sites of superoxide production in stimulated neutrophils.

In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.

Alkaline Phosphatase↗
PubMed1997

Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects.

To test whether a major contribution of airways epithelial ion transport to lung defense reflects the regulation of airway surface liquid (ASL) ionic composition, we measured ASL composition using the filter paper technique. On nasal surfaces, the Cl- concentration (approximately 125 meq/liter) was similar to plasma, but the Na+ concentration (approximately 110 meq/liter) was below plasma, and K+ concentration (approximately 30 meq/liter) above plasma. The resting ASL osmolarity [2(Na+ + K+); 277 meq/liter] approximated isotonicity. There were no detectable differences between cystic fibrosis (CF) and normal subjects. In the lower airways, the Na+ concentrations were 80-85 meq/liter, K+ levels approximately 15 meq/liter, and Cl- concentrations 75-80 meq/liter. Measurements of Na+ activity with Na(+)-selective electrodes and osmolality with freezing point depression yielded values consistent with the monovalent cation measurements. Like the nasal surfaces, no differences in cations were detected between CF, normal, or chronic bronchitis subjects. The tracheobronchial ASL hypotonicity was hypothesized to reflect collection-induced gland secretion, a speculation consistent with observations in which induction of nasal gland secretion produced hypotonic secretions. We conclude that there are no significant differences in ASL ion concentrations between CF, normal, and chronic bronchitis subjects and, because ASL ion concentrations exceed values consistent with defensin activity, the failure of CF lung defense may reflect predominantly factors other than salt-dependent defensins.

Adult↗
PubMed1997

Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation.

Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4 beta-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O2-) release. Immunofluorescent studies revealed that cofilin was present throughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H2O2), a product of the O2-/H2O2-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H2O2 production observed under certain conditions.

Actin Depolymerizing Factors↗
PubMed1997

Efficient immunocytochemical labeling of leukocyte microtubules with FluoroNanogold: an important tool for correlative microscopy.

We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.

Animals↗
PubMed1996

A method for co-localization of tubular lysosomes and microtubules in macrophages: fluorescence microscopy of individual cells.

Routinely used procedures for chemical fixation often fail to preserve delicate membrane-bounded tubular structures in a variety of cell types. Fixation procedures commonly employed in immunocytochemical studies for localization of structural proteins, such as those found in cytoskeletal elements, may also degrade these tubular structures. Here we describe a procedure that preserves the elaborate tubular lysosome system found in stimulated macrophages and allows the subsequent immunofluorescence localization of microtubules in the same cells. Use of this methodology permits the assessment of the spatial relationship between tubular lysosomes and microtubules in macrophages.

Animals↗
PubMed1995

Solubilization of glycosyl-phosphatidylinositol-anchored proteins in quiescent and stimulated neutrophils.

In human neutrophils, alkaline phosphatase (AlkPase), a low-affinity receptor for IgG (FcRIIIB), and complement decay accelerating factor (DAF) are glycosyl-phosphatidylinositol (GPI)-anchored proteins. Varying greatly in biological function these three integral membrane proteins exhibit regulated cell surface expression in neutrophils. Defined by their common membrane-linkage motif, AlkPase, FcRIIIB, and DAF can be released from the lipid bilayer by the action of phosphatidylinositol-specific phospholipase C and are relatively resistant to low temperature extraction with Triton X-100 (TX-100). In this study we show that neutrophil AlkPase, FcRIII, and DAF display differential extractibility; they are relatively insensitive to TX-100 solubilization at 4 degrees C, but are readily extracted with TX-100 at 37 degrees C or by the detergent octyl glucoside at 4 degrees C. The differential extractibility of these GPI-anchored proteins is the same in unstimulated cells, where these proteins exist primarily in an intracellular pool, and stimulated cells, where they are expressed principally at the cell surface. However, no differential extraction effect is observed with two neutrophil transmembrane proteins, complement receptor 1 (CD35, CR1) and MHC Class I in either stimulated or unstimulated cells.

Adult↗
PubMed1995

The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view.

The NADPH oxidase complex catalyzes the formation of superoxide (O2.-) in phagocytic leukocytes. This paper reviews recent advances in our understanding of this enzyme system. Recent studies have defined conditions for reconstitution of this enzymatic activity with purified proteins in a cell-free system. The role of the individual proteins that make up the active complex, their regulation and the effects of mutations in these proteins are discussed. While these studies represent major achievements, it is clear from cytochemical investigations that additional levels of complexity exist in the modulation of the NADPH oxidase complex in vivo. A major role for cytochemical analysis in understanding the cell biological aspects of the generation of reactive oxygen species is discussed.

Amino Acid Sequence↗
PubMed1995

Bone pate repair of the eroded incus--five years on.

Seventy-four patients undergoing bone pate repair of the long process of the incus as part of a tympanoplasty are presented. Mean follow-up time was 66 months (range 12-143 months). Hearing results are averaged over the frequencies 500, 1000, 2000 and 4000 Hz. Thirty-two per cent of the patients had hearing thresholds better than 30 dB pre-operatively. A significant improvement in hearing was seen (p < 0.001). The data were also analysed to assess the effect of the pre-operative state of the tympanic membrane on the final result. The pre-operative condition of the tympanic membrane was either perforated (35 ears) or atrophic (33 ears). Six tympanic membranes were intact but these were excluded from the final results. No difference in hearing result was found between patients who had an ossicular repair in combination with either a perforated tympanic membrane repair or an atrophic membrane repair. Average hearing gain in the perforated group was 13 dB, in the atrophic group 9 dB and in the intact group 7 dB.

Adolescent↗
PubMed1995

Stimulus-dependent alterations in macrophage microtubules: increased tubulin polymerization and detyrosination.

Murine macrophage microtubules are very dynamic. The majority of the microtubules (approximately 80%) exist in a rapidly depolymerizing pool (t1/2 approximately 30 seconds). The remaining 20% of the microtubules are in a more slowly depolymerizing pool (t1/2 approximately 7 minutes). Macrophage microtubules are responsive to cell stimulation with phorbol esters; upon cell stimulation there is a rapid increase in total microtubule polymer and number. In addition there is stimulus-induced detyrosination of alpha-tubulin in macrophage microtubules that is rapid and essentially complete, occurring in all microtubules rather than a subset of microtubules. Detyrosination of the macrophage microtubules in response to phorbol esters does not confer increased stability to these microtubules, since treated cells have nocodazole-induced depolymerization kinetics similar to that in non-stimulated macrophages. Regrowth of microtubules following washout of nocodazole is also rapid. Interestingly the regrown microtubules are initially in the tyrosinated form even in the presence of phorbol ester. These experiments provide in vivo support for the model that detyrosination of alpha-tubulin occurs in polymeric tubulin while retyrosination occurs in dimeric tubulin. Macrophage microtubules also demonstrate an unusually rapid response to extracellular stimuli and thus provide a unique model system in which to examine signal transduction events and modulation of microtubules.

Animals↗
PubMed1995

The microtubule cytoskeleton in human phagocytic leukocytes is a highly dynamic structure.

The microtubule cytoskeleton of human leukocytes has been difficult to study, in part, due to the lack of a reliable protocol for the indirect immunofluorescence staining of microtubules in these cells. We report here the development of a simple and reliable immunocytochemical labeling protocol for the examination of microtubules in leukocytes including monocytes, neutrophils, and eosinophils. The dynamic properties of microtubules in both monocytes and neutrophils were examined by indirect immunofluorescence staining of cells following exposure to nocodazole. Nocodazole-induced depolymerization is extremely rapid in both cell types, as is the regrowth of microtubules following removal of the nocodazole. Rapid reorganization of the microtubule cytoskeleton was also observed in neutrophils undergoing chemotactic stimulation. Bundling of microtubules was observed in both monocytes and neutrophils isolated from patients undergoing taxol infusion chemotherapy. The taxol-induced bundles were transient in nature as they were absent from samples collected 48 h following the completion of the taxol infusion. These results demonstrate the unique dynamic properties of leukocyte microtubules and indicate that they can be altered in vivo. The development of this staining protocol should allow for the further analysis of leukocyte microtubules as related to the normal functional response of these cells and form the basis for correlating alterations in microtubule dynamics with the effects of taxol on leukocyte function.

Drug-Related Side Effects and Adverse Reactions↗
PubMed1994

Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections.

We present a new application for the use of small immunogold particles (approximately 1.4-nm diameter) for ultrastructural immunocytochemistry. These small gold particles have been used on ultra-thin cryosections in conjunction with a silver enhancement procedure that does not degrade ultrastructural detail. We have used the human neutrophil as a model system, in which known protein markers of two different cytoplasmic granules were localized, in the development of this procedure. The 1.4-nm immunogold particles coupled with silver enhancement yield intense labeling for localization of lactoferrin, a marker for the specific granules, and myeloperoxidase, a marker for the azurophil granules. Double labeling in which one antigen was visualized with 1.4-nm gold and silver enhancement and a second antigen was detected with colloidal gold-IgG on the same ultra-thin cryosection was successfully achieved. We also show that 1.4-nm diameter immunogold particles penetrate into cryosectioned neutrophils to a greater extent than 5-nm or 10-nm immunogold probes. These results show that small immunogold particles, along with silver enhancement, are a useful addition to the immunolabeling methods available for use with ultra-thin cryosections.

Cytoplasmic Granules↗