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J S Reynolds

Publications and source records attributed to J S Reynolds.

16 recordsLinked to original sources
PubMed2001

Hyperthyroidism increases the risk of ozone-induced lung toxicity in rats.

The risk of lung injury from ozone exposure has been well documented. It is also known that various factors may significantly influence the susceptibility of animals to the toxic effects of ozone. In the present study, we investigated the possibility that hyperthyroidism might be associated with increases in ozone-induced pulmonary toxicity. To create a hyperthyroid condition, mature male Sprague--Dawley rats were given injections of thyroxine (dose range: 0.1 to 1 mg/kg body wt daily for 7 days). Control rats received vehicle injections. The animals were then exposed to air or ozone (dose range: 0.5 to 3 ppm for 3 h). At 18 h postexposure, bronchoalveolar lavage fluid and cells were harvested. In hyperthyroid animals, ozone exposure was associated with three- to sixfold increases in bronchoalveolar lavage fluid lactate dehydrogenase activities and albumin levels as well as the number of polymorphonuclear leukocytes harvested by bronchoalveolar lavage above levels observed in ozone-exposed control rats. Additional results from the present study suggest that these thyroid hormone-linked effects cannot be fully explained by differences in whole-body metabolic rate or changes in the inhaled dose of ozone. These findings indicate that the risk of ozone-induced lung toxicity is substantially increased in a hyperthyroid state and suggest that the susceptibility of the lung to damage from ozone exposure may be significantly influenced by individual thyroid hormone status.

Albumins↗
PubMed2000

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association.

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).

Amino Acid Substitution↗
PubMed2000

Molecular modelling and site-directed mutagenesis of the inositol 1,3,4,5-tetrakisphosphate-binding pleckstrin homology domain from the Ras GTPase-activating protein GAP1IP4BP.

GAP1(IP4BP) is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P(4)]. We have studied Ins(1,3,4,5)P(4) binding to GAP1(IP4BP), and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P(4) binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P(4) binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P(4) has shown that the binding site is located in a partially buried pocket between the beta 1/beta 2- and beta 3/beta 4-loops. Many of the residues involved in the binding are conserved in GAP1(IP4BP). Therefore we generated a model of the PH domain of GAP1(IP4BP) in complex with Ins(1,3,4,5)P(4) based on the Btk-Ins(1,3,4,5)P(4) complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P(4), indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.

Amino Acid Sequence↗
PubMed2000

Pharmacokinetics of ICG and HPPH-car for the detection of normal and tumor tissue using fluorescence, near-infrared reflectance imaging: a case study.

We present in vivo fluorescent, near-infrared (NIR), reflectance images of indocyanine green (ICG) and carotene-conjugated 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH-car) to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. Following intravenous administration of 1.0 mg kg-1 ICG or 0.3 mg kg-1 HPPH-car into the canine, a 25 mW, 778 nm or 70 mW, 660 nm laser diode beam, expanded by a diverging lens to approximately 4 cm in diameter, illuminated the surface of the mammary tissue. Successfully propagating to the tissue surface, ICG or HPPH-car fluorescence generated from within the tissue was collected by an image-intensified, charge-coupled device camera fitted with an 830 or 710 nm bandpass interference filter. Upon collecting time-dependent fluorescence images at the tissue surface overlying both normal and diseased tissue volumes, and fitting these images to a pharmacokinetic model describing the uptake (wash-in) and release (wash-out) of fluorescent dye, the pharmacokinetics of fluorescent dye was spatially determined. Mapping the fluorescence intensity owing to ICG indicates that the dye acts as a blood pool or blood persistent agent, for the model parameters show no difference in the ICG uptake rates between normal and diseased tissue regions. The wash-out of ICG was delayed for up to 72 h after intravenous injection in tissue volumes associated with disease, because ICG fluorescence was still detected in the diseased tissue 72 h after injection. In contrast, HPPH-car pharmacokinetics illustrated active uptake into diseased tissues, perhaps owing to the overexpression of LDL receptors associated with the malignant cells. HPPH-car fluorescence was not discernable after 24 h. This work illustrates the ability to monitor the pharmacokinetic delivery of NIR fluorescent dyes within tissue volumes as great as 0.5-1 cm from the tissue surface in order to differentiate normal from diseased tissue volumes on the basis of parameters obtained from the pharmacokinetic models.

Adenocarcinoma↗
PubMed2000

Effect of ozone treatment on airway reactivity and epithelium-derived relaxing factor in guinea pigs.

Ozone (O(3)) is toxic to respiratory epithelium and causes airway inflammation and hyperreactivity. To evaluate the role of the epithelium in the development of hyperreactivity, we examined in guinea pigs the effects of inhaled O(3) (3 ppm for 1 h; 0-24 h after exposure) on 1) reactivity to inhaled methacholine (MCh), 2) reactivity of the isolated, perfused trachea (IPT) to MCh, 3) epithelium-derived relaxing factor (EpDRF)-mediated relaxations of IPT induced by mucosal hyperosmolar solutions, 4) neurogenic contraction and relaxation responses, 5) transepithelial potential difference, and 6) microscopic analysis of nitrotyrosine immunofluorescence, substance P fiber density, and tracheal morphology. At 0 h, O(3) caused hyperreactivity to inhaled MCh and mucosally but not serosally applied MCh in IPT (only in the presence of the epithelium) and a decrease in transepithelial potential difference. Inhibition of EpDRF-induced relaxation responses occurred at 2 h. All of these changes returned to control by 12 to 18 h. O(3) had no effect on neurogenic responses. Nitrotyrosine immunofluorescence appeared in the trachea at 0 h in detached epithelial cell ghosts and in intrapulmonary airways by 6 h. Substance P fiber density was elevated in smooth muscle at 0 and 18 h but not in epithelium or lamina propria of intrapulmonary and extrapulmonary bronchi. Loss of cilia and mucosubstances in the mucosa occurred at 0 h; the epithelium became markedly attenuated over 12 to 24 h. A reversible increase in epithelial permeability and a decrease in EpDRF production may contribute to O(3)-induced hyperreactivity to MCh.

Animals↗
PubMed1999

Tissue-specific expression and endogenous subcellular distribution of the inositol 1,3,4,5-tetrakisphosphate-binding proteins GAP1(IP4BP) and GAP1(m).

GAP1(IP4BP) and GAP1(m) belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1(IP4BP) is primarily localised to the plasma membrane, whereas GAP1(m) appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1(m) and several Ca2+ store markers, consistent with the lack of co-localisation between GAP1(m) and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1(m) does not reside at a site where it could regulate the ability of InsP4 to release intracellular Ca2+. As GAP1(m) is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.

Animals↗
PubMed1999

Imaging of spontaneous canine mammary tumors using fluorescent contrast agents.

We present near-infrared frequency-domain photon migration imaging for the lifetime sensitive detection and localization of exogenous fluorescent contrast agents within tissue-simulating phantoms and actual tissues. We employ intensity-modulated excitation light that is expanded and delivered to the surface of a tissue or tissue-simulating phantom. The intensity-modulated fluorescence generated from within the volume propagates to the surface and is collected using a gain-modulated image-intensified charge-coupled device camera. From the spatial values of modulation amplitude and phase of the detected fluorescent light, micromolar volumes of diethylthiatricarbocyanine iodide (tau = 1.17 ns) and indocyanine green (ICG) (tau = 0.58 ns) embedded 1.0 cm deep in a tissue phantom are localized and discriminated on the basis of their lifetime differences. To demonstrate the utility of frequency-domain fluorescent measurements for imaging disease, we image the fluorescence emitted from the surface of in vivo and ex vivo canine mammary gland tissues containing lesions with preferential uptake of ICG. Pathology confirms the ability to detect spontaneous mammary tumors and regional lymph nodes amidst normal mammary tissue and fat as deep as 1.5 cm from the tissue surface.

Animals↗
PubMed1998

Structural and functional analysis of the putative inositol 1,3,4, 5-tetrakisphosphate receptors GAP1(IP4BP) and GAP1(m).

Previously we have purified and cloned a high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4)-binding protein which, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), we have termed GAP1(IP4BP). Here we show that expressed full-length GAP1(IP4BP) binds Ins(1,3,4, 5)P4 with an affinity and specificity similar to that of the originally purified protein, a binding activity which is dependent on a functional PH/Btk domain. Furthermore, we highlight a fundamental distinction between GAP1(IP4BP) and its homologue GAP1(m), namely that both proteins function as Ras GAPs but only GAP1(IP4BP) displays Rap GAP activity.

Base Sequence↗
PubMed1998

Fluorescence lifetime spectroscopic imaging with measurements of photon migration.

Frequency-domain measurements of photon migration are coupled with a model of fluorescence generation and propagation in order to develop a method for reconstructing maps of fluorescent properties within interior tissue volumes from exterior measurements at the air-tissue interface. Simulation results confirm the feasibility of optical imaging through the use of exogenously administered contrast agents on the basis of fluorophore decay kinetics and yield. Experimental measurements using single-pixel and multipixel devices illustrate that the contrast owing to exogenous fluorescence exceeds that owing to absorption or scattering caused by endogenous chromophores or tissue structure and owing to absorption caused by exogenous contrast agents.

Diagnostic Imaging↗
PubMed1997

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1IP4BP and GAP1m result from the GAP1IP4BP PH domain directing plasma membrane targeting.

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.

Animals↗
PubMed1997

Fluorescence and absorption contrast mechanisms for biomedical optical imaging using frequency-domain techniques.

The ability to optically image or detect diseased tissue volumes located deep within tissues depends upon the degree of contrast provided by differences in local optical properties. In this report, we show that the exogenous contrast offered by fluorescent compounds is superior to that provided by nonfluorescing, light-absorbing compounds when time-dependent measurements are employed. In addition, we show that the induced contrast is not only moderated by the preferential uptake of fluorescent agents into diseased tissue volumes of interest but also by the fluorescent optical properties and the fluorescence dynamics in the specific tissue volume. Using tissue phantom studies, we demonstrated experimentally that near-infrared-absorbing and fluorescent dyes such as indocyanine green can provide detection of diseased tissue volumes from fluorescence measurements made at the periphery of tissue when there is perfect, 100-fold and 10-fold partitioning in diseased tissues over that in surrounding normal tissues. Experimental results of common laser dyes show the contrast is also mediated by the quantum yield and lifetime parameters that may be dependent upon the local tissue environment.

Diagnosis↗
PubMed1997

Method to produce American Thoracic Society flow-time waveforms using a mechanical pump.

The American Thoracic Society (ATS) recently adopted a new set of 26 standard flow-time waveforms for use in testing both diagnostic and monitoring devices. Some of these waveforms have a higher frequency content than present in the ATS-24 standard volume-time waveforms, which, when produced by a mechanical pump, may result in a pump flow output that is less than the desired flow due to gas compression losses within the pump. To investigate the effects of gas compression, a mechanical pump was used to generate the necessary flows to test mini-Wright and Assess peak expiratory flow (PEF) meters. Flow output from the pump was measured by two different independent methods, a pneumotachometer and a method based on piston displacement and pressure measured within the pump. Measuring output flow based on piston displacement and pressure has been validated using a pneumotachometer and mini-Wright PEF meter, and found to accurately measure pump output. This method introduces less resistance (lower back-pressure) and dead space volume than using a pneumotachometer in series with the meter under test. Pump output flow was found to be lower than the desired flow both with the mini-Wright and Assess meters (for waveform No. 26, PEFs 7.1 and 10.9% lower, respectively). To compensate for losses due to gas compression, we have developed a method of deriving new input waveforms, which, when used to drive a commercially available mechanical pump, accurately and reliably produces the 26 ATS flow-time waveforms, even those with the fastest rise-times.

Evaluation Studies as Topic↗
PubMedDate not supplied

Multipixel techniques for frequency-domain photon migration imaging.

The ability to map interior optical properties of a highly scattering medium from exterior measurements of light propagation is afforded by optical tomography. In this communication, we describe the problem of optical tomography, the techniques of photon migration measurements necessary to accomplish it, and the development of multipixel measurements for rapid collection of optical signals. These multipixel measurements are shown to provide detection of contrast owing to the optical properties of absorption and fluorescence associated with dye-laden heterogeneities embedded in a tissue-like scattering medium. From these rapid measurements, successful reconstruction of an interior optical property map may now be possible with clinically realistic data acquisition times. Applications for the technology arise for biomedical optical imaging for the in vivo detection of disease and the diagnosis of tissue (bio-) chemistry.

Absorption↗