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Biomedical subjects

R L Patterson

Publications and source records attributed to R L Patterson.

At least 19 recordsLinked to original sources
PubMed2007

TRPC channels: integrators of multiple cellular signals.

TRPC channels are ubiquitously expressed among cell types and mediate signals in response to phospholipase C (PLC)-coupled receptors. TRPC channels function as integrators of multiple signals resulting from receptor-induced PLC activation, which catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 depletes Ca2+ stores and TRPC3 channels can be activated by store-depletion. InsP3 also activates the InsP3 receptor, which may undergo direct interactions with the TRPC3 channel, perhaps mediating store-dependence. The other PLC product, DAG, has a direct non-PKC-dependent activating role on TRPC3 channels likely by direct binding. DAG also has profound effects on the TRPC3 channel through PKC. Thus PKC is a powerful inhibitor of most TRPC channels and DAG is a dual regulator of the TRPC3 channel. PLC-mediated DAG results in rapid channel opening followed later by a slower DAG-induced PKC-mediated deactivation of the channel. The decreased level of PIP2 from PLC activation also has an important modifying action on TRPC3 channels. Thus, the TRPC3 channel and PLCgamma form an intermolecular PH domain that has high specificity for binding PIP2. This interaction allows the channel to be retained within the plasma membrane, a further operational control factor for TRPC3. As nonselective cation channels, TRPC channel opening results in the entry of both Na+ and Ca2+ ions. Thus, while they may mediate Ca2+ entry signals, TRPC channels are also powerful modifiers of membrane potential.

Animals↗
PubMed2001

Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels.

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.

Adenosine↗
PubMed2000

Ca2+ entry mediated by store depletion, S-nitrosylation, and TRP3 channels. Comparison of coupling and function.

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. SOC-mediated Ca(2+) entry is complex and may reflect more than one type of channel and coupling mechanism. To assess such possible divergence the function and coupling of SOCs was compared with two other distinct yet related Ca(2+) entry mechanisms. SOC coupling in DDT(1)MF-2 smooth muscle cells was prevented by the permeant inositol 1,4,5-trisphosphate (InsP(3)) receptor blockers, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C. In contrast, Ca(2+) entry induced by S-nitrosylation and potentiated by store depletion (Ma, H-T., Favre, C. J., Patterson, R. L., Stone, M. R., and Gill, D. L. (1999) J. Biol. Chem. 274, 35318-35324) was unaffected by 2-APB, suggesting that this entry mechanism is independent of InsP(3) receptors. The cycloalkyl lactamimide, MDL-12, 330A (MDL), prevented SOC activation (IC(50) 10 micrometer) and similarly completely blocked S-nitrosylation-mediated Ca(2+) entry. Ca(2+) entry mediated by the TRP3 channel stably expressed in HEK293 cells was activated by phospholipase C-coupled receptors but independent of Ca(2+) store depletion (Ma, H.-T., Patterson, R. L., van Rossum, D. B., Birnbaumer, L., Mikoshiba, K., and Gill, D. L. (2000) Science 287, 1647-1651). Receptor-induced TRP3 activation was 2-APB-sensitive and fully blocked by MDL. Direct stimulation of TRP3 channels by the permeant diacylglycerol derivative, 1-oleoyl-2-acetyl-sn-glycerol, was not blocked by 2-APB, but was again prevented by MDL. The results indicate that although the activation and coupling processes for each of the three entry mechanisms are distinct, sensitivity to MDL is a feature shared by all three mechanisms, suggesting there may be a common structural feature in the channels themselves or an associated regulatory component.

Animals↗
PubMed2000

Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels.

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.

Actins↗
PubMed2000

Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells.

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.

Animals↗
PubMed1999

Ca(2+) entry activated by S-nitrosylation. Relationship to store-operated ca(2+) entry.

The coupling between Ca(2+) pools and store-operated Ca(2+) entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca(2+) entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca(2+) pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T. , and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT(1)MF-2 smooth muscle cells and DC-3F fibroblasts, Ca(2+) entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca(2+) removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca(2+) entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca(2+) entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba(2+) entry and sensitivity to blockade by La(3+) were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca(2+) entry.

Animals↗
PubMed1999

Store-operated Ca2+ entry: evidence for a secretion-like coupling model.

The elusive coupling between endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) "store-operated" Ca2+ entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca2+ entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca2+ entry channels to Ca2+ store depletion. Coupling is concluded to be mediated by a physical "secretion-like" mechanism involving close but reversible interactions between the ER and the PM.

Actins↗
PubMed1997

The involvement of dopamine D2 receptors, but not D3 or D4 receptors, in the rewarding effect of brain stimulation in the rat.

To identify the subtype of dopamine receptors critically involved in the rewarding effect of brain stimulation, four dopamine antagonists were intracranially injected in 25 rats. The importance of dopamine D1 receptors had been demonstrated previously by using SCH 23390, a highly selective D1 antagonist. Rats were implanted with electrodes into the medial forebrain bundle and cannulae into either one of the following structures: the nucleus accumbens, the vicinity of the islands of Calleja, or the ventral tegmental area, all ipsilateral to the electrodes. The animals were trained to press a bar for electrical stimulation, and the frequency-response functions were plotted before and after injection of each dopamine antagonist through the cannulae. Raclopride and haloperidol, which have high affinities for D2 receptors, reduced the rewarding effect after injection into any one of the three cannula sites. Neither (+)-UH232, a selective D3 antagonist, nor clozapine, a D4 antagonist, influenced the rewarding effect. The results suggest that dopamine D2, but not D3 or D4, receptors are critically involved in producing the rewarding effect of brain stimulation.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1995

Irradiation-induced off-odour in chicken and its possible control.

1. Volatiles isolated from irradiated raw chicken were analysed by gas chromatography (GC) in conjunction with olfactory assessment of the effluent carrier gas to locate compounds with strong smells. 2. Sixteen odours of differing intensities were registered, some, but not others, coinciding with recognisable GC peaks. Identifications were made on the basis of retention data, mass spectrometric information and odour quality agreement. 3. Dimethyltrisulphide was found to be the most potent and obnoxious compound (foul gas, sulphurous), followed by cis-3- and trans-6-nonenals (soapy), oct-1-en-3-one (mushroom) and bis(methylthio-)methane (foul). With the exception of oct-1-en-3-one, none of these compounds has been reported before in irradiated raw chicken. 4. alpha-Tocopherol and ascorbic acid induce stability in tissues in vivo and post mortem. Chickens were reared on diets supplemented with high concentrations (800 mg/kg food) of each of these vitamins. Yields of irradiation volatiles from the tissues of these birds were very much reduced, compared to yields from similar tissues from birds fed unsupplemented diets. 5. Concomitantly with the reduced yield of volatiles, less odour was associated with the samples when analysed by GC-olfactory analysis. 6. The use of enhanced concentrations of the two vitamins in combination in the diet of poultry may provide a means of controlling development of off-odour in irradiated raw chicken, thus improving acceptability to the consumer.

Animal Feed↗
PubMed1992

Sounds of swallowing following total laryngectomy.

Accelerometer transduced sounds of swallowing in total laryngectomees did not show acoustical differences for liquid vs paste swallows, as are found in normals. Compared with normal swallows, tongue propulsion of the bolus in laryngectomee swallows occurred closer in time to a distinctive spectral change associated with bolus flow into the esophagus. Interpretation stressed the lack of mechanical traction from laryngeal elevation contributing to pharyngoesophageal sphincter opening, and the increased role of tongue propulsion in laryngectomee swallows.

Adult↗
PubMed1990

Review of current techniques for the verification of the species origin of meat.

An overview of developments that have occurred in meat species identification over the last decade is presented. It starts by noting the different requirements for speciation techniques over the period, describes the complex nature of meat in terms of chemical composition and shows how the chain of events from slaughter to retail gives rise to opportunities for deliberate adulteration or innocent contamination. The limitations of techniques such as electrophoresis and isoelectrofocusing are pointed out where the analysis of mixed meats is concerned; attention then focuses on the range of techniques based on antigen-antibody interactions: agar gel immunodiffusion, counter immuno-electrophoresis and enzyme-linked immunosorbent assay (ELISA) in three formats. The choice of analyte is discussed, firstly for the analyses of raw meat materials and secondly, for heat-processed meat products. In the first example, blood serum proteins are used almost exclusively despite the limitation that their presence does not necessarily denote the presence of the corresponding muscle tissue (meat). For cooked products, a new range of antisera are necessary, based on thermally stable components derived from the tissues. By using different formats of ELISA, it is demonstrated that different responses can be obtained for heat-processed meat versus processed offal, and that determination of a species meat content in a cooked mixed meat product is possible. Techniques for improving the specificity and performance of antisera are discussed briefly, with the future introduction of thermally stable, muscle-specific monoclonal antisera being seen as the way forward.

Animals↗
PubMed1990

Interpreting the sounds of swallowing: fluid flow through the cricopharyngeus.

The sounds of swallowing in normal subjects were recorded by means of a miniature accelerometer on the throat, simultaneously with videofluoroscopy of a modified barium swallow. Consistent correspondence was found between a rapid change in the acoustic spectrum of the accelerometer signal (the appearance of a region of energy prominence above 1,000 Hz) and bolus flow through the cricopharyngeus.

Adult↗
PubMed1985

Binding of steroid hormones and anabolic agents to bovine sex-hormone binding globulin.

Binding of some selected steroids and anabolic agents to bovine sex-hormone binding globulin (SHBG) was investigated. SHBG binding affinities, relative to the reference hormone 5 alpha-dihydrotestosterone, were estimated for the compounds. The results demonstrate that binding of steroid hormones to SHBG is facilitated by the 17 beta-hydroxyl group, possibly involving hydrogen binding, and by the methyl group at C-19 of the steroid moiety. Structural modifications at C-17 of a steroid molecule involving esterification, epimerization or reduction of the 17 beta-hydroxyl group, or introduction of a bulky 17 alpha group have the effect of decreasing the SHBG binding affinity of the steroid molecule.

Androstenedione↗