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R J Hohman

Publications and source records attributed to R J Hohman.

At least 19 recordsLinked to original sources
PubMed1999

In situ amplification using universal energy transfer-labeled primers.

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used.

Cell Line↗
PubMed1999

Detection of telomerase activity utilizing energy transfer primers: comparison with gel- and ELISA-based detection.

We have developed a closed-tube format telomeric repeat amplification protocol (TRAP) assay for direct quantification of telomerase activity within the PCR vessel. The assay utilizes energy transfer (ET) primers, which emit fluorescence only upon incorporation into PCR products. This novel ET primer system (Amplifluor primers) has major advantages over existing detection methods because it eliminates the need for post-PCR processing and thus reduces greatly the risk of carryover contamination and the time required for the sample analysis. The assay is as sensitive, specific and quantitative as the polyacrylamide gel-based or ELISA-based TRAP assay.

DNA Primers↗
PubMed1997

A closed tube format for amplification and detection of DNA based on energy transfer.

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.

Base Sequence↗
PubMed1994

Hydrogen peroxide effects on rat mast cell function.

Oxidant exposure of the airway mucosa may play a significant role in the pathophysiology of asthma and allergic rhinitis. Mast cells play an important role in asthma, and oxidant exposure has been reported to cause direct mast cell degranulation as well as augment immunoglobulin E (IgE)-mediated responses in vivo. H2O2 is an oxidant generated by inflammatory cells and by the interaction of ozone with lipids or aqueous solutions. In this study, the RBL-2H3 mast cell line was used to investigate the ability of H2O2 to induce mast cell responses as well as to effect mast cell responses to IgE and the calcium ionophore A23187. Although cytotoxicity of RBL-2H3 cells at the membrane level was not observed with any concentration of H2O2, DNA damage resulted from exposure to 0.2 and 2.0 mM H2O2, and cell proliferation was inhibited by 0.075-0.2 mM H2O2. RBL cell prostaglandin D2 generation was enhanced after 60- and 120-min exposure to 0.2-20 mM H2O2. Direct serotonin release required 120-min exposures to 2.0 mM and 60-min exposures to 20 mM H2O2. However, degranulation responses induced by either IgE or A23178 were diminished after exposure to 0.2-2.0 mM H2O2. Lesser amounts (0.005-0.02 mM) had no effect on mast cell function. In summary, H2O2-induced responses of RBL cells, as well as modification of responses to IgE and A23187, occurred only at high concentrations of H2O2, which also induced both intracellular damage and inhibition of cell proliferation. Concentrations of H2O2 more likely to be physiologically relevant had no effect on mast cell responses or cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Orthovanadate induces translocation of phospholipase C-gamma 1 and -gamma 2 in permeabilized mast cells.

Rapid activation of phospholipase C (PLC) with a resultant increase in phosphatidylinositol hydrolysis occurs after aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells. We previously described an increase in PLC activity associated with the particulate fraction of rat basophilic leukemia (RBL) cells after Fc epsilon RI aggregation, and this redistribution of enzyme activity correlated with an increase in immunoreactivity of the gamma 1 isozyme of PLC in the particulate fraction by Western blot analysis (J. Immunol. 148:2194-2200, 1992). We now report that the tyrosine phosphatase inhibitor orthovanadate mimics Fc epsilon RI-mediated activation of PLC-gamma 1 in RBL cells after permeabilization with Staphylococcus aureus alpha-toxin. Orthovanadate treatment of permeabilized cells induced: 1) a large increase in phosphoinositide hydrolysis in endogenously labeled cells; 2) an increase in PLC activity associated with the particulate fraction; and 3) an increase in immunoreactivity of PLC-gamma 1 in Western blots of the particulate fraction. In addition, incubation of RBL cells with either oligomeric IgE or orthovanadate results in the translocation of PLC-gamma 2 from the cytosol to the particulate fraction. All of the above effects were qualitatively similar to those seen after Fc epsilon RI aggregation. These data suggest that translocation and activation of PLC in mast cells are controlled by tyrosine phosphorylation of either the enzyme itself or some regulatory component. The equilibrium can be shifted to the phosphorylated state during either receptor-mediated activation of a tyrosine kinase or by blockade of dephosphorylation.

Animals↗
PubMed1993

Substance P, calcitonin gene-related peptide, and vasoactive intestinal peptide increase in nasal secretions after allergen challenge in atopic patients.

BACKGROUND: There is suggestive evidence that neuropeptides participate in allergic reactions. Substance P (SP) and calcitonin gene-related peptide (CGRP) are released by sensory nerves, whereas vasoactive intestinal peptide (VIP) is released mainly by parasympathetic nerves. Both sets of nerves are thought to be stimulated by allergic inflammation. The aim of this study was to assess nasal secretions to determine whether SP, CGRP, and VIP were increased after allergen challenge. METHODS: Eight patients with allergic rhinitis were challenged nasally with 1 mg histamine or increasing doses of allergen. Nasal lavages were collected into a cocktail of protease inhibitors in order to restrict neuropeptide degradation. Radioimmunoassay for SP, CGRP, and VIP were performed on each sample. RESULTS: All patients had immediate clinical reactions to both histamine and allergen challenges, and seven patients experienced a later allergic reaction. After histamine challenge, SP and CGRP did not increase significantly above baseline in the nasal lavages, whereas VIP did (p < 0.02). In contrast, SP, CGRP, and VIP all significantly increased immediately after allergen challenge and returned to baseline within 2 hours. At the clinical peak of the late allergic reaction, SP, but not CGRP or VIP, was increased slightly but significantly (p < 0.01). CONCLUSIONS: Thus SP, CGRP, and VIP are found in nasal secretions after allergen challenge, which confirms that neuropeptides are released in human beings during allergic reactions. The selective stimulation of VIP secretion by histamine challenge suggests that histamine-induced cholinergic reflexes induce the release of VIP. These data support the suggestion that neuropeptides may be partly responsible for some of the nasal symptoms of allergy.

Adult↗
PubMed1993

Distribution of secretory leukoprotease inhibitor in the human nasal airway.

Secretory leukoprotease inhibitor (SLPI) is a secreted glandular protein thought to regulate elastase activity and, more recently, to inhibit both mast cell chymase activity and histamine release from mast cells. To begin to examine the possible role of SLPI in humans, we determined the distribution of SLPI in the human nasal mucosa and quantitated the functional activity of SLPI in nasal lavage fluid. Immunochemical staining of the nasal mucosa revealed intense, selective immunoreactivity in the serous cells of the submucosal glands. The level of SLPI in nasal secretions was measured by enzyme immunoassay. In control subjects (n = 8), the level of SLPI in nasal lavage fluid (NLF) after saline challenge (baseline level) was 2.5 +/- 0.5 micrograms/ml, accounting for 3.3 +/- 0.6% of total protein in nasal secretions. After methacholine (MCh) and histamine (HIST) challenge, the level of SLPI increased to 7.0 +/- 1.4 and 6.1 +/- 1.6 micrograms/ml, respectively (both p < 0.05). In atopic patients (n = 8), the level of SLPI after MCh and HIST challenge increased from a baseline level of 7.6 +/- 2.0 micrograms/ml to 22.1 +/- 6.4 and 25.2 +/- 10.5 micrograms/ml, respectively. After allergen challenge, the concentration of SLPI increased significantly in atopic patients, whereas there was no increase in the level of SLPI in control subjects. Western blot analysis of MCh-induced nasal secretions revealed a single band with a molecular weight of 12 kD, the same as recombinant SLPI.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Human nasal mucosal neutral endopeptidase (NEP): location, quantitation, and secretion.

Neutral endopeptidase (E.C.3.4.24.11, enkephalinase, NEP) is a potentially important enzyme capable of regulating the activity of neuropeptides released in the respiratory mucosa. In order to confirm the existence of NEP in the human respiratory mucosa, inferior nasal turbinate mucosae obtained at surgery and nasal secretions induced by topical provocations with methacholine, histamine, and allergen were analyzed for: (1) NEP activity (pmol product/min/ml) by enzymatic degradation of [3H]leu-enkephalin, (2) the presence of NEP-immunoreactive material by Western blot analysis, and (3) cellular localization of NEP distribution by immunohistochemistry. NEP activity in human nasal secretions obtained after normal saline challenge was 0.15 +/- 0.06 pmol/min/ml. Secretion increased to 0.86 +/- 0.26 pmol/min/ml after methacholine provocation and 1.69 +/- 0.74 pmol/min/ml after histamine provocation. The increase in NEP activity in methacholine-induced secretions was prevented by atropine (0.13 +/- 0.06 pmol/min/ml). After methacholine, histamine, and antigen nasal provocation, the kinetics of NEP appearance correlated more closely to the glandular marker, lactoferrin, than with the vascular markers albumin and IgG. In homogenates of nasal mucosa, the membrane fraction contained significantly more NEP on a per mg protein basis than did the soluble fraction (227.6 +/- 50.52 versus 9.61 +/- 3.18 pmol/min/mg protein, respectively, P < 0.01, n = 6). NEP in the membrane fraction was detected as a single band migrating at 97 kD on Western blots using antibodies specific for NEP and the common acute lymphoblastic leukemia antigen (CALLA). Immunoreactive NEP was localized to serous cells of the submucosal glands, epithelial cells, and endothelial and myoepithelial cells of small vessels. Staining for NEP in the serous cells was of the same intensity as that in epithelial cells. These results indicate that 97 kD NEP-immunoreactive material exists in discrete locations in the nasal mucosa, including the epithelium, serous cells of the submucosal glands, and vessel walls, and that NEP activity is detected as a minor component in nasal secretions enriched by glandular products. In addition to the modulating functions of NEP on neuropeptide-mediated activities on vessels and glands, it is possible that NEP in secretions plays a role in regulating mucosal responses to luminal neuropeptides or other as yet uncharacterized NEP substrates.

Blotting, Western↗
PubMed1992

Evidence for GTP-binding protein involvement in the tyrosine phosphorylation of the T cell receptor zeta chain.

The zeta subunit of the T cell receptor (TCR) is a prominent substrate for a TCR-activated tyrosine kinase. Tyrosine phosphorylation of the zeta subunit in response to antibody-mediated receptor cross-linking was synergized in permeabilized T cells by either of two non-hydrolyzable GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) or guanosine 5'-[beta, gamma-imido]triphosphate Gpp(NH)p. ATP analogues did not significantly affect antibody-induced tyrosine phosphorylation. Unlike the GTP analogues, the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP beta S) did not enhance phosphorylation of zeta. The effect induced by the GTP analogues required TCR occupancy and was independent of protein kinase C. Taken together these observations implicate a GTP-binding protein in the modulation of TCR-induced tyrosine phosphorylation.

Animals↗
PubMed1992

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.

Animals↗
PubMed1992

The effect of the immunophilin ligands rapamycin and FK506 on proliferation of mast cells and other hematopoietic cell lines.

The immunosuppressive drugs FK506 and cyclosporin A have an identical spectrum of activities with respect to IgE receptor (Fc epsilon RI)-mediated exocytosis from mast cells and T cell receptor-mediated transcription of IL-2. These findings suggest a common step in receptor-mediated signal transduction leading to exocytosis and transcription and imply that immunosuppressive drugs target specific signal transduction pathways, rather than specific cell types. This hypothesis is supported by studies on the effect of rapamycin on IL-3 dependent proliferation of the rodent mast cell line PT18. Rapamycin inhibits proliferation of PT18 cells, achieving a plateau of 80% inhibition at 1 nM. This inhibition is prevented in a competitive manner by FK506, a structural analogue of rapamycin. Proliferation of rat basophilic leukemia cells and WEHI-3 cells was also inhibited, at doses comparable to those shown previously to inhibit IL-2-dependent proliferation of cytotoxic T lymphocyte line (CTLL) cells. In contrast, proliferation of A-431 cells, a epidermoid cell line, was not affected by rapamycin. DNA histograms indicate that complexes formed between the rapamycin-FK506-binding protein (FKBP) and rapamycin arrest-proliferating PT18 cells in the G0/G1-phase. It is concluded that FKBP-rapamycin complexes may inhibit proliferative signals emanating from IL-3 receptors, resulting in growth arrest of cytokine-dependent, hematopoietic cells.

Animals↗
PubMed1991

Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription.

Investigations of the actions and interactions of the immunophilin ligands FK506, cyclosporin A (CsA), rapamycin, and 506BD suggest that complexes of FK506 with an FK506-binding protein or of CsA with a cyclophilin (CsA-binding protein) inhibit the T-cell receptor-mediated signal transduction that results in the transcription of interleukin 2. Now we report an identical spectrum of activities of FK506, CsA, rapamycin, and 506BD on IgE receptor-mediated signal transduction that results in exocytosis of secretory granules from the rat basophilic leukemia cell line RBL-2H3, a mast cell model. Both FK506 and CsA inhibit receptor-mediated exocytosis (CsA IC50 = 200 nM; FK506 IC50 = 2 nM) without affecting early receptor-associated events (hydrolysis of phosphatidylinositol, synthesis and release of eicosanoids, uptake of Ca2+). In contrast, rapamycin and 506BD, which share common structural elements with FK506, by themselves have no effect on IgE receptor-mediated exocytosis. Both compounds, however, prevent inhibition by FK506 but not by CsA. Affinity chromatography with FK506, CsA, and rapamycin matrices indicates that the same set of immunophilins present in RBL-2H3 cells have been found in Jurkat T cells and calf thymus; however, the relative amounts of these proteins differ in the two cell types. These results suggest the existence of a common step in cytoplasmic signaling in T cells and mast cells that may be part of a general signaling mechanism.

Animals↗
PubMed1990

IgE receptor-mediated phosphatidylinositol hydrolysis and exocytosis from rat basophilic leukemia cells are independent of extracellular Ca2+ in a hypotonic buffer containing a high concentration of K+.

In isotonic buffer, IgE receptor-mediated exocytosis from rat basophilic leukemia cells is dependent on extracellular Ca2+, with half-maximal degranulation requiring 0.4 mM Ca2+. No significant exocytosis occurs in the absence of extracellular Ca2+. This absolute requirement for Ca2+ is eliminated by suspending the cells in a hypotonic buffer containing 60 to 80 mM K+; Na+ cannot substitute for K+. Optimal Ca2(+)-independent exocytosis occurs in a buffer containing 20 mM dipotassium Pipes, pH 7.1, 40 mM KCl, 5 mM glucose, 7 mM Mg acetate, 0.1% BSA, and 1 mM EGTA. The cells maintain this Ca2(+)-independent exocytosis even if they are preincubated with 1 mM EGTA for 40 min at 37 degrees C before triggering. Exocytosis is eliminated as isotonicity is approached by adding sucrose, NaCl, KCl, or potassium glutamate to the buffer. Quin 2 fluorescence measurements reveal only a very small rise in [Ca2+]i when the cells are triggered in hypotonic buffer in the absence of extracellular Ca2+ and the presence of 1 mM EGTA. In isotonic buffer, degranulation does not occur under conditions that lead to such a small rise in [Ca2+]i. Sustained IgE receptor-mediated phosphatidylinositol hydrolysis, which is also Ca2+ dependent in isotonic buffer, becomes independent of Ca2+ in the hypotonic buffer. In fact, the rate of phosphatidylinositol hydrolysis in hypotonic buffer in the absence of Ca2+ (and presence of 1 mM EGTA) is twice that observed in isotonic buffer in the presence of 1 mM Ca2+. These data show that in hypotonic buffer, the requirement of IgE receptor-mediated PI hydrolysis for extracellular Ca2+ is eliminated, and degranulation proceeds with a [Ca2+]i of 0.1 microM, the baseline level of [Ca2+]i found in resting cells. These results are consistent with the hypothesis that, in isotonic buffer, the Ca2+ requirement for mast cell degranulation is for the generation of second messengers via hydrolysis of membrane phosphatidylinositols.

Animals↗
PubMed1990

Cyclosporin A inhibits degranulation of rat basophilic leukemia cells and human basophils. Inhibition of mediator release without affecting PI hydrolysis or Ca2+ fluxes.

Cyclosporin A (CSA) inhibits IgE receptor-mediated exocytosis from rat basophilic leukemia (RBL) cells and human peripheral blood basophils in a dose-dependent manner over the therapeutic range of CSA concentrations achieved in vivo. Half-maximal inhibition was observed at 0.2 micrograms/ml CSA. The effect of CSA on several biochemical parameters involved in receptor-mediated activation of RBL cells was examined. Maximum inhibition of secretion occurred when CSA was added 5 min before activation, and inhibition was nearly maximum when the drug was added 2 min before the cells were triggered. The same results were observed when RBL cells were stimulated with A23187, a calcium ionophore. These results suggest a mechanism other than inhibition of protein synthesis is involved. Inhibition by CSA of release by either secretagogue persisted, even if CSA was removed from the buffer before the cells were triggered. No inhibition was observed of either receptor-mediated phosphatidylinositol hydrolysis, 45Ca2+ uptake, or the rise in the intracellular concentration of free Ca2+ under the same conditions that produced greater than 80% inhibition of serotonin release. These results demonstrate that the early events in signal transduction are not affected, and suggest that the intracellular target for CSA participates in a later stage of exocytosis. Furthermore, the data suggest that CSA suppresses cells other than T lymphocytes and predict that patients on CSA therapy may have altered response to allergens.

Animals↗
PubMed1990

Cyclosporin A: new insights for cell biologists and biochemists.

Cyclosporin A (CSA) is well known for its potent immunosuppressive properties. Until recently, most of the research on the mechanism of action of CSA focused on its effects on cytokine transcription by T lymphocytes. However, CSA inhibits a variety of other cellular functions. An intracellular CSA-binding protein, called cyclophilin, has been purified and characterized. This protein is found in nearly all mammalian cells, which suggests that it is involved in highly conserved cellular functions. The current concept is that CSA mediates its effect via cyclophilin. Cyclophilin is actually a peptidyl-prolyl cis-trans isomerase (PPIase), an enzyme proposed to catalyze protein folding. Because the binding of CSA to cyclophilin/PPIase in vitro inhibits the isomerase activity, it is thought that this may account for the inhibitory effects of CSA on the cellular functions described above. To add to the puzzle, a new immunosuppressive drug, FK-506, has recently been shown to bind to an intracellular protein similar to, but distinct from, cyclophilin. The FK-506 binding protein also has a PPIase activity, and this activity is inhibited by FK-506. These data are consistent with the hypothesis that CSA and FK-506 mediate their effects on cellular functions by inhibiting an isomerase activity required for protein folding. This hypothesis poses several interesting questions. For example, how is this protein folding step involved in such diverse cellular functions as gene transcription and granule exocytosis? Verification of the role of CSA and PPIase in cellular functions awaits the identification of the substrates for the isomerases.

Amino Acid Isomerases↗
PubMed1988

Assessment of IgE-receptor function through measurement of hydrolysis of membrane inositol phospholipids. New insights on the phenomena of biphasic antigen concentration-response curves and desensitization.

Previous studies have shown that hydrolysis of membrane inositol phospholipids in rat basophilic leukemia (RBL-2H3) cells depended on the rate and extent of the aggregation of receptors of IgE. This response was used as an experimental probe to study the role of IgE receptors in initiating stimulatory and inhibitory processes within the cell. The response was amplified markedly by increasing the concentration of external Ca2+ from 0 to 1 mM, but the concentration required to support half-maximal response varied from less than 0.1 mM for the most potent cross-linking reagent, DNP24BSA (24 molecules of DNP attached to 1 molecule of BSA) to 0.5 mM for the least potent reagent, aggregated OVA. The dependency of phosphoinositide hydrolysis on external Ca2+ was reduced to zero once hydrolysis of inositol phospholipids was underway but secretion of histamine remained totally dependent on the presence of 0.5 to 1 mM external Ca2+. The stimulatory response persisted as long as receptors remained aggregated but it was modulated by a biochemical process, possibly the activation of protein kinase C, that targeted specifically aggregated receptors, or an associated protein. For example, when cells had become desensitized to high concentrations of one Ag, a normal response could be evoked with a second Ag. Also cells that had become desensitized could be reactivated by permeabilizing the cells. Interestingly, bell-shaped Ag dose-response curves, which were characteristic for both the phosphoinositide and secretory responses, were transformed to sigmoid-shaped curves once cells were permeabilized and dialyzed.

Animals↗