Several aspects of the behavior of antibodies at substrate-supported planar membranes have been c... more Several aspects of the behavior of antibodies at substrate-supported planar membranes have been characterized with quantitative fluorescence microscopy: (1) very slow rotational mobilities of an anti-dinitrophenyl monoclonal antibody associated with solid-like phospholipid Langmuir-Blodgett monolayers have been measured with polarized fluorescence photobleaching recovery; (2) the equilibrium and kinetics of an anti-dinitrophenyl Fab at phospholipid Langmuir-Blodgett monolayers have been characterized with total internal reflection fluorescence microscopy; and (3) the apparent association constants of a panel of anti-dinitrophenyl mouse IgGl antibodies with substrate-supported planar membranes containing purified and reconstituted mouse IgG Fc receptors have been measured with total internal reflection fluorescence microscopy.
Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional recon... more Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.
BACKGROUND: Laparoscopic hysterectomies comprise a large proportion of all hysterectomies in the ... more BACKGROUND: Laparoscopic hysterectomies comprise a large proportion of all hysterectomies in the United States. Procedures completed under regional anesthesia pose a number of benefits to patients, but laparoscopic hysterectomies traditionally have been performed under general anesthesia. We describe a case of total laparoscopic hysterectomy under epidural anesthesia with the patient fully awake. CASE: A 51-year-old woman with abnormal uterine bleeding underwent an uncomplicated total laparoscopic hysterectomy, bilateral salpingectomy, and excision of endometriosis. The procedure was completed under epidural anesthesia without intravenous sedation or systemic narcotics. Pneumoperitoneum with a pressure of 12 mm Hg and Trendelenburg to 15° allowed for adequate visualization. Anesthesia was achieved with midthoracic and low lumbar epidural catheters. Bilevel positive airway pressure was used for augmentation of respiratory function. CONCLUSION: With a committed patient, adequate plann...
Several aspects of the behavior of antibodies at substrate-supported planar membranes have been c... more Several aspects of the behavior of antibodies at substrate-supported planar membranes have been characterized with quantitative fluorescence microscopy: (1) very slow rotational mobilities of an anti-dinitrophenyl monoclonal antibody associated with solid-like phospholipid Langmuir-Blodgett monolayers have been measured with polarized fluorescence photobleaching recovery; (2) the equilibrium and kinetics of an anti-dinitrophenyl Fab at phospholipid Langmuir-Blodgett monolayers have been characterized with total internal reflection fluorescence microscopy; and (3) the apparent association constants of a panel of anti-dinitrophenyl mouse IgGl antibodies with substrate-supported planar membranes containing purified and reconstituted mouse IgG Fc receptors have been measured with total internal reflection fluorescence microscopy.
We have used HPLC techniques to investigate the effects of gp120 upon inositol phosphate turnover... more We have used HPLC techniques to investigate the effects of gp120 upon inositol phosphate turnover in Jurkat E6‐1 CD4+ T‐cells, to pursue previous reports that this viral coat protein: (a) inhibits receptor‐activated inositol phosphate release; (b) stimulates basal inositol phosphate release; (c) inhibits inositol polyphosphate 5‐phosphatase. Treatment of cells with up to 10 μg/ml gp120 from between 10 min and 24 h was without effect upon inositol phosphate turnover in both basal cells, and in C305 and OKT3 stimulated cells. This is the first report that biologically competent gp120 does not affect any aspect of inositol phosphate turnover in either basal or receptor‐activated lymphocytes.
Norepinephrine has long been known to stimulate the pulsatile and preovulatory release of LH-rele... more Norepinephrine has long been known to stimulate the pulsatile and preovulatory release of LH-releasing hormone (LHRH). In vivo and in vitro studies indicate that these effects are mediated primarily through α1-adrenergic receptors (α1-ARs). With the immortalized hypothalamic LHRH neurons, we have found that α1-adrenergic agents directly stimulate the secretion of LHRH in a dose-dependent manner. Ligand binding and RNA studies demonstrate that the GT1 cells contain both α1A- and α1B-ARs. Competition binding experiments show that approximately 75% of the binding is due toα 1B-ARs; the remainder is made up ofα 1A-ARs. Receptor activation leads to stimulation of PLC. PLCβ1 and PLCβ3 are expressed in GT1 neurons, and these PLCs are probably responsible for the release of diacylglycerol and IP as well as the increase in intracellular calcium. The mobilization of cytoplasmic calcium is sufficient to stimulate cytosolic PLA2 (cPLA2) and release arachidonic acid. A dissection of the contribu...
Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional recon... more Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.
The binding of five different mouse monoclonal anti‐dinitrophenyl (DNP) IgGI monomers, in the pre... more The binding of five different mouse monoclonal anti‐dinitrophenyl (DNP) IgGI monomers, in the presence and absence of DNP‐glycine, to FcγRII on the mouse macrophage‐like cell line J774 has been investigated. Membrane association constants were estimated using a competitive radioimmunoassay in which the ability of the IgGl monomers to block the binding of 125I‐labelled Fabs of the anti‐FcγRII monoclonal antibody 2.4G2 was monitored. The IgGl‐FcγRII association constants ranged from (8 ± 1) × 104 M−1 to (6 ± 2) × 105 M−1. No large differences in the IgGl‐FcγRII association constants were measured for different IgG1 antibodies or for any single IgG1 antibody in the presence and absence of DNP‐glycine.
Several aspects of the behavior of antibodies at substrate-supported planar membranes have been c... more Several aspects of the behavior of antibodies at substrate-supported planar membranes have been characterized with quantitative fluorescence microscopy: (1) very slow rotational mobilities of an anti-dinitrophenyl monoclonal antibody associated with solid-like phospholipid Langmuir-Blodgett monolayers have been measured with polarized fluorescence photobleaching recovery; (2) the equilibrium and kinetics of an anti-dinitrophenyl Fab at phospholipid Langmuir-Blodgett monolayers have been characterized with total internal reflection fluorescence microscopy; and (3) the apparent association constants of a panel of anti-dinitrophenyl mouse IgGl antibodies with substrate-supported planar membranes containing purified and reconstituted mouse IgG Fc receptors have been measured with total internal reflection fluorescence microscopy.
Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional recon... more Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.
BACKGROUND: Laparoscopic hysterectomies comprise a large proportion of all hysterectomies in the ... more BACKGROUND: Laparoscopic hysterectomies comprise a large proportion of all hysterectomies in the United States. Procedures completed under regional anesthesia pose a number of benefits to patients, but laparoscopic hysterectomies traditionally have been performed under general anesthesia. We describe a case of total laparoscopic hysterectomy under epidural anesthesia with the patient fully awake. CASE: A 51-year-old woman with abnormal uterine bleeding underwent an uncomplicated total laparoscopic hysterectomy, bilateral salpingectomy, and excision of endometriosis. The procedure was completed under epidural anesthesia without intravenous sedation or systemic narcotics. Pneumoperitoneum with a pressure of 12 mm Hg and Trendelenburg to 15° allowed for adequate visualization. Anesthesia was achieved with midthoracic and low lumbar epidural catheters. Bilevel positive airway pressure was used for augmentation of respiratory function. CONCLUSION: With a committed patient, adequate plann...
Several aspects of the behavior of antibodies at substrate-supported planar membranes have been c... more Several aspects of the behavior of antibodies at substrate-supported planar membranes have been characterized with quantitative fluorescence microscopy: (1) very slow rotational mobilities of an anti-dinitrophenyl monoclonal antibody associated with solid-like phospholipid Langmuir-Blodgett monolayers have been measured with polarized fluorescence photobleaching recovery; (2) the equilibrium and kinetics of an anti-dinitrophenyl Fab at phospholipid Langmuir-Blodgett monolayers have been characterized with total internal reflection fluorescence microscopy; and (3) the apparent association constants of a panel of anti-dinitrophenyl mouse IgGl antibodies with substrate-supported planar membranes containing purified and reconstituted mouse IgG Fc receptors have been measured with total internal reflection fluorescence microscopy.
We have used HPLC techniques to investigate the effects of gp120 upon inositol phosphate turnover... more We have used HPLC techniques to investigate the effects of gp120 upon inositol phosphate turnover in Jurkat E6‐1 CD4+ T‐cells, to pursue previous reports that this viral coat protein: (a) inhibits receptor‐activated inositol phosphate release; (b) stimulates basal inositol phosphate release; (c) inhibits inositol polyphosphate 5‐phosphatase. Treatment of cells with up to 10 μg/ml gp120 from between 10 min and 24 h was without effect upon inositol phosphate turnover in both basal cells, and in C305 and OKT3 stimulated cells. This is the first report that biologically competent gp120 does not affect any aspect of inositol phosphate turnover in either basal or receptor‐activated lymphocytes.
Norepinephrine has long been known to stimulate the pulsatile and preovulatory release of LH-rele... more Norepinephrine has long been known to stimulate the pulsatile and preovulatory release of LH-releasing hormone (LHRH). In vivo and in vitro studies indicate that these effects are mediated primarily through α1-adrenergic receptors (α1-ARs). With the immortalized hypothalamic LHRH neurons, we have found that α1-adrenergic agents directly stimulate the secretion of LHRH in a dose-dependent manner. Ligand binding and RNA studies demonstrate that the GT1 cells contain both α1A- and α1B-ARs. Competition binding experiments show that approximately 75% of the binding is due toα 1B-ARs; the remainder is made up ofα 1A-ARs. Receptor activation leads to stimulation of PLC. PLCβ1 and PLCβ3 are expressed in GT1 neurons, and these PLCs are probably responsible for the release of diacylglycerol and IP as well as the increase in intracellular calcium. The mobilization of cytoplasmic calcium is sufficient to stimulate cytosolic PLA2 (cPLA2) and release arachidonic acid. A dissection of the contribu...
Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional recon... more Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.
The binding of five different mouse monoclonal anti‐dinitrophenyl (DNP) IgGI monomers, in the pre... more The binding of five different mouse monoclonal anti‐dinitrophenyl (DNP) IgGI monomers, in the presence and absence of DNP‐glycine, to FcγRII on the mouse macrophage‐like cell line J774 has been investigated. Membrane association constants were estimated using a competitive radioimmunoassay in which the ability of the IgGl monomers to block the binding of 125I‐labelled Fabs of the anti‐FcγRII monoclonal antibody 2.4G2 was monitored. The IgGl‐FcγRII association constants ranged from (8 ± 1) × 104 M−1 to (6 ± 2) × 105 M−1. No large differences in the IgGl‐FcγRII association constants were measured for different IgG1 antibodies or for any single IgG1 antibody in the presence and absence of DNP‐glycine.
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Papers by Martina Sumner