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J W Parce

Publications and source records attributed to J W Parce.

At least 19 recordsLinked to original sources
PubMed2001

Synchronized cyclic capillary electrophoresis using channels arranged in a triangle and low voltages.

Synchronized cyclic capillary electrophoresis (SCCE) makes use of a closed loop separation channel by which the same sample can be separated during many cycles. This enables the repeated use of the same voltage for separations such that a high total voltage, and thus high efficiency, is obtained for the synchronized components. This can be accomplished by using any type of polygon geometry for the separation channel; and calculations of the available field and number of connections needed for polygons from 3 to 5 sides are presented. Triangular designs have the advantage of using the lowest number of wells. Such designs are described, with two additional features compared to that of earlier work: 1. voltage connections that are much shallower than the separation channel, to reduce losses and dispersion at the intersections; and 2. corners that are narrower than the separation channels to reduce dispersion in the turns. Experimental data is presented for the separation of a mixture of amino acids, and for a DNA separation in a polymeric sieving matrix. The DNA separation is most sensitive to the corner dispersion problem, which reduces the observed efficiency for that separation.

Amino Acids↗
PubMed2001

Multiport flow-control system for lab-on-a-chip microfluidic devices.

A multiport system suitable for pressure control on a lab-on-a-chip microfluidic device is described. An algorithm and a strategy for calculating pressures were developed to control the flow from multiple reservoirs for the microfluidic devices. Dye mixing and enzyme assay titration experiments were performed using pressure-driven flow only. Results show a good linear response over two orders of dynamic range.

Algorithms↗
PubMed2000

Electrokinetically controlled microfluidic analysis systems.

Electrokinetic forces are emerging as a powerful means to drive microfluidic systems with flow channel cross-sectional dimensions in the tens of micrometers and flow rates in the nanoliter per second range. These systems provide many advantages such as improved analysis speed, improved reproducibility, greatly reduced reagent consumption, and the ability to perform multiple operations in an integrated fashion. Planar microfabrication methods are used to make these analysis chips in materials such as glass or polymers. Many applications of this technology have been demonstrated, such as DNA separations, enzyme assays, immunoassays, and PCR amplification integrated with microfluidic assays. Further development of this technology is expected to yield higher levels of functionality of sample throughput on a single microfluidic analysis chip.

Chemistry Techniques, Analytical↗
PubMed2000

Test tube's end.

Explore the source record for details and available documents.

Genetic Techniques↗
PubMed1994

Microfabrication in silicon microphysiometry.

Over the past 5 years, microphysiometry has proved an effective means for detecting physiological changes in cultured cells, particularly as a functional assay for the activation of many cellular receptors. To demonstrate the clinical relevance of this method, we have used it to detect bacterial antibiotic sensitivity and to discriminate between bacteriostatic and bacteriocidal concentrations. The light-addressable potentiometric sensor, upon which microphysiometry is based, is well suited for structural manipulations based on photolithography and micromachining, and we have begun to take advantage of this capability. We present results from a research instrument with eight separate assay channels on a 5-cm2 chip. We discuss the planned evolution of the technology toward high-through-put instruments and instruments capable of performing single-cell measurements.

Biosensing Techniques↗
PubMed1993

GM-CSF triggers a rapid, glucose dependent extracellular acidification by TF-1 cells: evidence for sodium/proton antiporter and PKC mediated activation of acid production.

The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic "cell capsule" that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 18 h PMA treatment. Short-term treatment with PMA (1-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cells, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.

Benzoquinones↗
PubMed1993

Cholinergic stimulation of the Na+/K+ adenosine triphosphatase as revealed by microphysiometry.

The activation of a wide range of cellular receptors has been detected previously using a novel instrument, the microphysiometer. In this study microphysiometry was used to monitor the basal and cholinergic-stimulated activity of the Na+/K+ adenosine triphosphatase (ATPase) (the Na+/K+ pump) in the human rhabdomyosarcoma cell line TE671. Manipulations of Na+/K+ ATPase activity with ouabain or removal of extracellular K+ revealed that this ion pump was responsible for 8.8 +/- 0.7% of the total cellular energy utilization by those cells as monitored by the production of acid metabolites. Activation of the pump after a period of inhibition transiently increased the acidification rate above baseline, corresponding to increases in intracellular [Na+] ([Na+]i) occurring while the pump was off. The amplitude of this transient was a function of the total [Na+]i excursion in the absence of pump activity, which in turn depended on the duration of pump inhibition and the Na+ influx rate. Manipulations of the mode of energy metabolism in these cells by changes of the carbon substrate and use of metabolic inhibitors revealed that, unlike some other cells studied, the Na+/K+ ATPase in TE671 cells does not depend on any one mode of metabolism for its adenosine triphosphate source. Stimulation of cholinergic receptors in these cells with carbachol activated the Na+/K+ ATPase via an increase in [Na+]i rather than a direct activation of the ATPase.

Biophysical Phenomena↗
PubMed1992

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Linking Reagents↗
PubMed1992

The cytosensor microphysiometer: biological applications of silicon technology.

A silicon-based device, dubbed a microphysiometer, can be used to detect and monitor the response of cells to a variety of chemical substances, especially ligands for specific plasma membrane receptors. The microphysiometer measures the rate of proton excretion from 10(4) to 10(6) cells. This article gives an overview of experiments currently being carried out with this instrument with emphasis on receptors with seven transmembrane helices and tyrosine kinase receptors. As a scientific instrument, the microphysiometer can be thought of as serving two distinct functions. In terms of detecting specific molecules, selected biological cells in this instrument serve as detectors and amplifiers. The microphysiometer can also investigate cell function and biochemistry. A major application of this instrument may prove to be screening for new receptor ligands. In this respect, the microphysiometer appears to offer significant advantages over other techniques.

Animals↗
PubMed1992

Antigen-specific stimulation of T cell extracellular acidification by MHC class II-peptide complexes.

A specific T cell response to a preformed complex of detergent-solubilized MHC class II molecule and cognate antigenic peptide was observed by monitoring the extracellular acidification. An increase in this rate was observed when the resting 4R3.9 T cell clone specific for the peptide fragment MBP(1-14) of myelin basic protein was exposed to preformed detergent-solubilized IAk-MBP(1-14)A4 complexes. MBP peptide alone, IAk alone, or complexes of IAs-proteolipid protein(139-151) and IAd-OVA(323-339), did not cause significant increases in the acidification rates of the MBP(1-14)-restricted 4R3.9 T cell clone. In addition, BW 5147 T lymphoma cells, which lack TCR, did not show any increase in rate when exposed to IAk-MBP(1-14)A4 complexes. Similar increases in acidification rate were observed in the presence of IL-2, anti-CD3 and anti-TCR antibodies. The enhanced acidification responses were blocked by genistein, a tyrosine kinase inhibitor.

Acids↗
PubMed1992

Biosensors based on the energy metabolism of living cells: the physical chemistry and cell biology of extracellular acidification.

The silicon microphysiometer is a biosensor-based instrument that detects changes in the physiological state of cultured living cells by monitoring the rate at which the cells excrete acidic products of metabolism. This paper discusses the chemical and biological factors that determine the performance and applications of such a system. Under typical culture conditions, extracellular acidification is dominated by the excretion of lactic and carbonic acids formed during the energy metabolism, using glucose and glutamine as carbon sources. The maintenance of transmembrane ionic gradients is an important use of energy, as is cell growth. The activation of cellular receptors usually causes transient or sustained increases in acidification rate. The energetic cost of generating second messengers is probably too small to account for either change, so events more distal to the receptor-activation process must be responsible. The opening of ion channels may cause the increases in some cases. In others, changes in intracellular pH and loose coupling between ATP hydrolysis and synthesis may be involved; models for these processes are presented.

Acids↗
PubMed1992

Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein.

The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo.

Fluorescence↗
PubMed1990

A new bifunctional spin-label suitable for saturation-transfer EPR studies of protein rotational motion.

A new bifunctional spin-label (BSL) has been synthesized that can be immobilized on the surface of proteins, allowing measurement of rotational motion of proteins by saturation-transfer electron paramagnetic resonance (STEPR). The spin-label contains a photoactivatable azido moiety, a cleavable disulfide, and a nitroxide spin with restricted mobility relative to the rest of the label. The label reacts with surface lysine residues modified with beta-mercaptopropionate. Bifunctional attachment is achieved by photoactivation of the azido group. Any spin-label that remains monofunctionally attached after photolysis is removed by reduction of the disulfide. Only bifunctionally attached BSL remains on the protein. Hemoglobin was used to test the utility of the BSL in STEPR by comparison with hemoglobin modified with maleimide spin-label (MSL), a commonly used standard for the STEPR technique. MSL is a monofunctional spin-label which is fortuitously immobilized by local protein structure within hemoglobin. The BSL labeling of hemoglobin did not significantly affect the quaternary structure of hemoglobin as determined by gel filtration chromatography. The conventional EPR spectra of the mono- and bifunctionally attached BSL-hemoglobin were similar to the MSL-hemoglobin spectrum, indicating that both forms of BSL were rigidly bound to hemoglobin. In contrast, the spectrum obtained by reaction of modified hemoglobin lysine residues with MSL indicated that these labels were highly mobile. The monofunctionally attached BSL was mobilized upon octyl glucoside addition whereas bifunctionally attached BSL was only slightly mobilized, suggesting that hydrophobic interactions immobilize the monofunctionally attached label on hemoglobin. The response of STEPR spectra of mono- and bifunctionally attached BSL-hemoglobin to changes in hemoglobin rotational correlation time was similar to the MSL-hemoglobin over the range of 10(-5)-10(-3) s. The spectra of bifunctionally attached BSL indicated slightly less motion than corresponding spectra for MSL or monofunctionally attached BSL. The new BSL is a good reporter of protein rotation and does not require unique protein structures for its immobilization on the protein. Thus, the BSL should be more generally applicable for STEPR studies of membrane protein rotation than existing monofunctional spin-labels.

Cross-Linking Reagents↗
PubMed1990

Dynamic nature of the quaternary structure of the vesicular stomatitis virus envelope glycoprotein.

The envelope glycoprotein (G protein) of vesicular stomatitis virus probably exists in the viral envelope as a trimer of identical subunits. Depending on the conditions of solubilization, G protein may dissociate into monomers. G protein solubilized with the detergent octyl glucoside was shown to exist as oligomeric forms by sedimentation velocity analysis and chemical cross-linking. G protein was modified with either fluorescein isothiocyanate or rhodamine isothiocyanate. Resonance energy transfer between fluorescein and rhodamine labels was observed upon mixing the two labeled G proteins in octyl glucoside. This result provided further evidence that G protein in octyl glucoside is oligomeric and indicated that the subunits are capable of exchange to form mixed oligomers. Resonance energy transfer was independent of G protein concentration in the range examined (10-80 nM) and was not observed when labeled G proteins were mixed with fluorescein or rhodamine that was not conjugated to protein. Resonance energy transfer decreased upon incorporation of G protein into Triton X-100, consistent with sedimentation velocity data that G protein in Triton X-100 is primarily monomeric. Kinetic analysis showed that the subunit exchange reaction had a half-time of about 3 min at 27 degrees C that was independent of G protein concentration. These data indicate that the exchange occurs through dissociation of G protein trimers into monomers and dimers followed by reassociation into timers. Thus, in octyl glucoside, G protein must exist as an equilibrium between monomers and oligomers. This implies that monomers are capable of self-assembly into trimers.

Centrifugation, Density Gradient↗
PubMed1990

A fluorometric method for determining the degree of biotinylation of proteins.

A method to determine the number of biotin moieties attached to a protein has been developed based on quenching the natural fluorescence of avidin or streptavidin by biotin. The assay consists of titrating the number of biotin combining sites on streptavidin/avidin before and after adding the biotinylated protein. With this method only those biotin moieties capable of binding to streptavidin/avidin are detected. The assay is simple and sensitive, requiring only 1-10 micrograms of biotinylated protein per determination.

Animals↗
PubMed1990

Continuous monitoring of receptor-mediated changes in the metabolic rates of living cells.

Activation of beta-adrenergic or muscarinic acetylcholine receptors expressed in transfected cells or epidermal growth factor receptors in human keratinocytes produces 15% to 200% changes in cellular metabolic rates. Changes in cell metabolism were monitored continuously with a previously described silicon-based microphysiometer that detects small changes in extracellular pH. The amplitude and kinetics of the metabolic changes depend upon several factors including pretreatment of the cells prior to receptor stimulation, the dose of hormone/neurotransmitter used, and the receptor complement of the cells. Responses are receptor specific; cells transfected with receptor genes respond only to the appropriate hormone/transmitter, whereas control (nontransfected) cells or cells transfected with different receptors exhibit no response. The specificity of the responses was further documented by using pharmacological antagonists. In Chinese hamster ovary (CHO) cells transfected with human beta 2-adrenergic receptors, isoproterenol produces a 20-60% increase in the rate of extracellular acidification with an EC50 of 4 nM, a response that is competitively antagonized by (-)-propranolol. The EC50 for the isoproterenol response is shifted from 4 nM to 100 nM in the presence of 3 nM (-)-propranolol. The kinetics of the metabolic response induced by beta-adrenergic receptor stimulation are markedly slower than those elicited by muscarinic receptor agonists. The maximal metabolic response in cells transfected with beta-adrenergic receptors peaks at approximately 12 min as compared with less than 30 sec in cells transfected with muscarinic receptors, perhaps reflecting activation of different second-messenger pathways. These findings illustrate an alternative means of studying cellular responses to hormones and neurotransmitters and suggest that metabolic changes will be generally useful for detecting the consequences of receptor-ligand interactions.

Animals↗