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R S Lewis

Publications and source records attributed to R S Lewis.

At least 19 recordsLinked to original sources
PubMed2007

Analysis of an introgressed Nicotiana tomentosa genomic region affecting leaf number and correlated traits in Nicotiana tabacum.

Germplasm from closely related diploid relatives of tobacco (Nicotiana tabacum L.) could be of value for continued genetic modification of this species and for mapping quantitative trait loci (QTLs). We examined near isogenic tobacco lines and hybrids differing for an introgressed genomic region from N. tomentosa Ruiz and Pavon designated as Many Leaves that exhibits a large influence on leaf number and correlated traits. Within a 'Red Russian' genetic background, the region acted in an additive to partially dominant fashion to delay flowering time, and increase leaf number, plant height, and green leaf yield. Evidence of epistasis was observed as the region affected these traits to varying degrees in diverse near isogenic hybrids. Fifteen amplified fragment length polymorphism (AFLP) markers of N. tomentosa origin were mapped within a single linkage group of 34.5 cM using a population of 207 BC(1)F(1) individuals segregating for Many Leaves. Composite interval mapping produced 2-LOD confidence intervals for likely QTL positions influencing leaf number (3.1 cM region), plant height (2.9 cM region), and days to flowering (3.3 cM region). These intervals were overlapping. Results demonstrate that genomic regions with large genetic effects can be transferred to tobacco from closely related diploid relatives, and that sufficient recombination within these regions may permit mapping of genes controlling quantitative traits. Materials and results described here may be useful in future research to gain insight on the genetic control of the transition from vegetative to reproductive development in Nicotiana.

Amplified Fragment Length Polymorphism Analysis↗
PubMed2005

Transfer of resistance to potato virus Y (PVY) from Nicotiana africana to Nicotiana tabacum: possible influence of tissue culture on the rate of introgression.

A disomic chromosome addition line of tobacco, Nicotiana tabacum L., was established previously that possesses a single chromosome pair from N. africana [Merxm. and Buttler]. This addition chromosome carries a gene that confers increased resistance to severe strains of potato virus Y (PVY). Methods to increase the probability of gene transfer from alien chromosomes to tobacco (2n=48) are desired. In the research described here, the PVY resistance gene was transferred to a tobacco chromosome from the N. africana addition chromosome in seven independent cases. One introgression event was obtained using conventional backcrossing of the disomic addition line to N. tabacum cv. Petite Havana, while the remaining six events were obtained using a scheme that involved exposure of explants of the addition line to tissue culture. Twenty-six derived 2n=48 individuals heterozygous for PVY resistance were found to exhibit 24 bivalents or 23 bivalents + 2 univalents at metaphase I. Ovular transmission rates for the PVY resistance factor ranged from 25% to 52%, while pollen transmission rates were much lower, ranging from 0 to 39%. Fifty-one random amplified polymorphic DNA (RAPD) markers specific for the intact addition chromosome were identified and used to characterize derived 2n=48/PVY-resistant genotypes. Variability was observed among these plants with respect to the total number of N. africana RAPD markers that were present, which is an indication that crossing over was occurring within each of the seven introgressed chromosome segments. A limited molecular marker-assisted backcrossing experiment allowed for selection of a 2n=48/PVY-resistant individual that possessed only 6 of the 51 original N. africana RAPD markers. In vitro culture is potentially a valuable system for increasing the rate of alien gene transfer in tobacco, and the successful transfer of PVY resistance from N. africana may allow for an increased level and range of resistance to this virus in tobacco.

Chromosomes, Plant↗
PubMed2004

Analysis of immobilized L-cysteine on polymers.

Recently, we reported that L-cysteine attached to polymeric biomaterials, without prior nitrosation, enhances the hemocompatibility of biomaterials via exploiting endogenous nitric oxide (NO). As part of the polymer optimization process to further enhance platelet inhibition, a kinetic model is being developed to predict the release rate of NO. A key model parameter is the immobilized concentration of L-cysteine. This article demonstrates how several chemiluminescence-based assays, previously utilized for measuring thiols in solution, were successfully adapted to quantify immobilized L-cysteine. The assays showed that the immobilized L-cysteine on the modified PET sample is within the range of 4.1 to 6.5 nmol/cm(2). An advantage of using the more successful chemiluminescence-based assay is that it can accurately measure molar concentrations of any thiol-containing compound with a detection limit in the pmol range. The major disadvantage is that L-cysteine must first be broken off of the polymer and released into solution prior to measurement, therefore leaving the sample unable to be reused. Other thiol-measuring techniques, such as fluorescence microscopy and X-ray photoelectron spectroscopy (XPS), were used to provide qualitative and semiquantitative analysis to substantiate the polymer development.

Cysteine↗
PubMed2003

Incorporation of tropical maize germplasm into inbred lines derived from temperate x temperate-adapted tropical line crosses: agronomic and molecular assessment.

Exotic maize ( Zea mays L.) germplasm may allow for increased flexibility and greater long-term progress from selection if it can be incorporated at high rates into U.S. breeding programs. Crosses were made between a temperate line, NC262A, and each of eight different lines consisting of 100% temperate-adapted tropical germplasm. Pedigree selection was used to generate a set of 148 F(5)S(2) lines that were evaluated in testcrosses with FR992/FR1064 in nine North Carolina environments. Several entries had grain yield, grain moisture content and standability that were comparable to three commercial checks. The best testcrosses outyielded the cross NC262A x FR992/FR1064 by 9.5 to 10.9%, suggesting that a significant amount of tropical germplasm was retained in these lines and that this germplasm combined well with the Stiff Stalk tester. Previous researchers had suggested that tropical alleles could be rapidly lost during inbreeding in populations derived from tropical x temperate bi-parental crosses, leading to the development of lines that possess significantly less than 50% tropical germplasm. F(5)S(5) sub-lines corresponding to the 14 best testcrosses were genotyped at 47 to 49 polymorphic simple sequence repeat (SSR) loci across all ten chromosomes to estimate the amount of tropical germplasm that was retained. The estimated genetic contribution from the tropical parent ranged from 32 to 70%, with the average being 49%. Only two of the 14 lines deviated significantly from a 50%-tropical/50%-temperate ratio, suggesting limited overall selection against germplasm from the tropical parents. These experiments collectively demonstrated that tropical maize germplasm can be incorporated at high rates into a temperate line via pedigree breeding methods in order to derive new inbred lines with acceptable agronomic performance.

Alleles↗
PubMed2003

Calcium oscillations in T-cells: mechanisms and consequences for gene expression.

[Ca(2+)](i) (intracellular Ca(2+) concentration) oscillations play a central role in the activation of T-lymphocytes by antigen. Oscillations in T-cells are absolutely dependent on Ca(2+) influx through store-operated CRAC channels (Ca(2+)-release-activated Ca(2+) channels), and evidence suggests that they arise from delayed interactions between these channels and Ca(2+) stores. Their potential functions have been explored by creating controlled [Ca(2+)](i) oscillations with pulses of Ca(2+) entry or pulses of Ins(1,4,5)P(3). Oscillations enhance both the efficiency and specificity of signalling through the Ca(2+)-dependent transcription factors nuclear factor of activated T-cells (NFAT), Oct/Oap and nuclear factor kappa B (NF kappa B) in ways that are consistent with each factor's Ca(2+) dependence and kinetics of activation and deactivation. These studies show how [Ca(2+)](i) oscillations may enhance signalling to the nucleus, and suggest a possible cellular mechanism for extracting information encoded in oscillation frequency.

Animals↗
PubMed2002

Polytype distribution in circumstellar silicon carbide.

The inferred crystallographic class of circumstellar silicon carbide based on astronomical infrared spectra is controversial. We have directly determined the polytype distribution of circumstellar SiC from transmission electron microscopy of presolar silicon carbide from the Murchison carbonaceous meteorite. Only two polytypes (of a possible several hundred) were observed: cubic 3C and hexagonal 2H silicon carbide and their intergrowths. We conclude that this structural simplicity is a direct consequence of the low pressures in circumstellar outflows and the corresponding low silicon carbide condensation temperatures.

Astronomical Phenomena↗
PubMed2001

Potentiation and inhibition of Ca(2+) release-activated Ca(2+) channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP(3) receptors.

1. The effects of the IP(3)-receptor antagonist 2-aminoethyldiphenyl borate (2-APB) on the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in Jurkat human T cells, DT40 chicken B cells and rat basophilic leukaemia (RBL) cells were examined. 2. 2-APB elicited both stimulatory and inhibitory effects on Ca(2+) influx through CRAC channels. At concentrations of 1-5 microM, 2-APB enhanced Ca(2+) entry in intact cells and increased I(CRAC) amplitude by up to fivefold. At levels > or = 10 microM, 2-APB caused a transient enhancement of I(CRAC) followed by inhibition. 3. 2-APB altered the kinetics of fast Ca(2+)-dependent inactivation of I(CRAC). At concentrations of 1-5 microM, 2-APB increased the rate of fast inactivation. In contrast, 2-APB at higher concentrations (> or = 10 microM) reduced or completely blocked inactivation. 4. 2-APB inhibited Ca(2+) efflux from mitochondria. 5. 2-APB inhibited I(CRAC) more potently when applied extracellularly than intracellularly. Furthermore, increased protonation of 2-APB at low pH did not affect potentiation or inhibition. Thus, 2-APB may have an extracellular site of action. 6. Neither I(CRAC) activation by passive store depletion nor the effects of 2-APB were altered by intracellular dialysis with 500 microg ml(-1) heparin. 7. I(CRAC) is present in wild-type as well as mutant DT40 B cells lacking all three IP(3) receptor isoforms. 2-APB also potentiates and inhibits I(CRAC) in both cell types, indicating that 2-APB exerts its effects independently of IP(3) receptors. 8. Our results show that CRAC channel activation does not require physical interaction with IP(3) receptors as proposed in the conformational coupling model. Potentiation of I(CRAC) by 2-APB may be a useful diagnostic feature for positive identification of putative CRAC channel genes, and provides a novel tool for exploring the physiological functions of store-operated channels.

Animals↗
PubMed2001

Effects of nitric oxide (NO) and soluble nucleoside triphosphate diphosphohydrolase (NTPDase) on inhibition of platelet deposition in vitro.

Vascular thrombosis is regulated via the release of several constituents from the vascular endothelium, including nucleoside triphosphate diphosphohydrolases (NTPDases or ectonucleotidases), nitric oxide (NO), and eicosanoids. Currently, it is unknown how these constituents interact in the inhibition of platelet aggregation and adhesion. To investigate the combined effects of NO and NTPDase on platelet deposition sequestration, an in vitro study was performed to compare inhibition of platelet deposition to a biomaterial by NO in the absence or presence of soluble NTPDase. Results of the platelet inhibition studies with NO and NTPDase conclusively show that the inhibitory effects of NTPDase and NO are additive. The platelet inhibitory potency in the presence of NO was enhanced by NTPDase in a dose-dependent manner, for a given NO exposure. This augmentation is independent of aspirin; the ability of NTPDase or NO alone to inhibit platelet deposition is also independent of aspirin. Clearly, NO and NTPDase independently contribute to platelet inhibition via different mechanisms. The inaction of NO on the activity of NTPDase confirmed that NO or reaction products in the presence of O(2) do not interact with NTPDase directly.

Acid Anhydride Hydrolases↗
PubMed2001

Calcium signaling mechanisms in T lymphocytes.

Elevation of intracellular free Ca(2+) is one of the key triggering signals for T-cell activation by antigen. A remarkable variety of Ca(2+) signals in T cells, ranging from infrequent spikes to sustained oscillations and plateaus, derives from the interactions of multiple Ca(2+) sources and sinks in the cell. Following engagement of the T cell receptor, intracellular channels (IP3 and ryanodine receptors) release Ca(2+) from intracellular stores, and by depleting the stores trigger prolonged Ca(2+) influx through store-operated Ca(2+) (CRAC) channels in the plasma membrane. The amplitude and dynamics of the Ca(2+) signal are shaped by several mechanisms, including K(+) channels and membrane potential, slow modulation of the plasma membrane Ca(2+)-ATPase, and mitochondria that buffer Ca(2+) and prevent the inactivation of CRAC channels. Ca(2+) signals have a number of downstream targets occurring on multiple time scales. At short times, Ca(2+) signals help to stabilize contacts between T cells and antigen-presenting cells through changes in motility and cytoskeletal reorganization. Over periods of minutes to hours, the amplitude, duration, and kinetic signature of Ca(2+) signals increase the efficiency and specificity of gene activation events. The complexity of Ca(2+) signals contains a wealth of information that may help to instruct lymphocytes to choose between alternate fates in response to antigenic stimulation.

Animals↗
PubMed2000

Mitochondrial control of calcium-channel gating: a mechanism for sustained signaling and transcriptional activation in T lymphocytes.

In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca(2+) signals. In many cells, mitochondria act as Ca(2+) buffers by taking up and releasing Ca(2+), but this simple buffering action by itself often cannot explain the organelle's effects on Ca(2+) signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca(2+) channels in T lymphocytes as a mechanism of mitochondrial Ca(2+) signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca(2+) stores causes sustained activation of the store-operated Ca(2+) current, I(CRAC) (CRAC, Ca(2+) release-activated Ca(2+)). Inhibition of mitochondrial Ca(2+) uptake by compounds that dissipate the intramitochondrial potential unmasks Ca(2+)-dependent inactivation of I(CRAC). Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca(2+) accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca(2+) uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca(2+)](i) elevation, indicating a functional link between mitochondrial regulation of I(CRAC) and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca(2+) channel activity and signal transmission from the plasma membrane to the nucleus.

Calcium Channels↗
PubMed2000

Influence of agonist, shear rate, and perfusion time on nitric oxide inhibition of platelet deposition.

Nitric oxide (NO) is a physiological species involved in inhibition of platelet adhesion and aggregation. A novel NO delivery device was utilized to quantitatively assess the effects of gaseous NO on platelet deposition to agonist-coated biomaterials in the presence of a platelet suspension. Platelet deposition was evaluated as a function of agonist (collagen, fibrinogen, or IgG), shear rate (250, 500, and 750 s(-1)), and perfusion time (5, 7.5, and 15 min). The minimal aqueous surface NO concentrations and fluxes necessary for significant inhibition of platelet deposition were quantified. Platelet deposition was completely inhibited at a gaseous NO exposure of 0.1 ppm, irrespective of the platelet agonist, shear rate, and perfusion time. The corresponding aqueous surface NO concentration was 0.09 nM at 250 s(-1) as predicted by a validated model. Surface fluxes ranged between 0.3 and 0.6 femtomoles cm(-2) s(-1). The results of this study are useful for establishing generalized guidelines (i.e., NO flux requirements in the presence of agonists, shear rate, and perfusion time) for the design and development of suitable biomaterials incorporating NO to reduce platelet deposition. Further studies incorporating blood, rather than platelet suspensions, are required to provide a more complete assessment of the required NO flux necessary to inhibit platelet deposition.

Adult↗
PubMed2000

Nitric oxide, superoxide, and peroxynitrite effects on the insulin secretion and viability of betaTC3 cells.

The onset of insulin-dependent diabetes mellitus (IDDM) is often associated with the infiltration of pancreatic cells by macrophages. Upon activation, macrophages release nitric oxide (NO) and superoxide (O2). These species or their reactive intermediates can be cytoxic, mutagenic, or carcinogenic. Previous studies have reported both positive and negative effects of extracellularly generated NO on insulin secretion and viability of pancreatic cells. Inherent problems of several previous studies assessing the effects of NO on insulin secretion include unsteady state NO concentration exposures and the generation of other potentially damaging species. In this study, these problems were eliminated by using a modified experimental system in which NO delivery was achieved via diffusion across a gas-permeable tube and O2 delivery was maintained using an enzymatic reaction. The delivery rates were constant, leading to steady state concentrations of NO and O2 in the experimental system. Based on reaction kinetics, a model was developed to predict NO, O2, and peroxynitrite (ONOO-) concentrations during the experiment. This study showed that NO, O2, and ONOO- at predicted concentrations as high as 2.8 microM, 0.25 microM, and 0.1 nM, respectively, do not affect the insulin secretion rates of betaTC3 pancreatic cells over short times.

Animals↗
PubMed1999

Nitric oxide inhibition of platelet deposition on biomaterials.

Platelet adhesion and aggregation restrict the clinical applicability of blood-contacting biomaterials. Nitric oxide (NO) is a simple biological molecule that may be incorporated into biomaterials to inhibit platelet deposition. The toxicity of NO at superphysiological levels necessitates the determination of aqueous NO concentrations and fluxes that effectively inhibit platelet deposition. In this study, a novel NO delivery device has been developed to study NO inhibition of platelet deposition in a dynamic in vitro environment. Gaseous NO was delivered via a semipermeable membrane to a radiolabeled platelet suspension perfusing a thin flow slit. The membrane was coated with a platelet-agonistic protein. Spatial NO flux and concentration profiles in the flow slit are predictable using a mathematical model. Platelet inhibition was essentially complete at 0.1 ppm gaseous NO exposure, corresponding to a surface concentration of 0.09 nM and surface fluxes between 0.3 and 0.6 femtomoles cm-2s-1. These threshold values of NO exposure for significant platelet inhibition were unchanged irrespective of the platelet agonist, perfusion times, or shear rates. At lower NO exposures (0.02 ppm), platelet inhibition was only partial with the degree of inhibition dependent on the nature of the agonistic protein. This study yields information useful towards the design and development of biomaterials incorporating NO for the reduction of platelet-biomaterial interactions.

Biocompatible Materials↗
PubMed1998

Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction.

BACKGROUND: Antigen-receptor interactions on lymphocytes result in local clustering of actin, receptors and signaling molecules into an asymmetric membrane structure termed a cap. Although actin polymerization is known to be required, the mechanisms underlying cap formation are unclear. We have studied the events underlying cap formation using mice bearing a null mutation in vav (vav-/-), a gene that encodes a guanine-nucleotide exchange factor for the GTPase Rac. RESULTS: Lymphocytes from vav-/- mice failed to form T-cell receptor caps following activation and had a defective actin cytoskeleton. The vav-/- T cells were deficient in interleukin-2 (IL-2) production and proliferation, and the peak of Ca2+ mobilization was reduced although of normal duration. Activation of Jun N-terminal kinase or stress-activated kinase (JNK or SAPK) and mitogen-activated protein kinase (MAPK) and the induction of the transcription factor NF-ATc1 and egr-1 genes was normal. Despite the reduced Ca2+ mobilization, translocation of cytoplasmic NF-ATc to the nucleus was normal, reflecting that the lower levels of Ca2+ in vav-/- cells were still sufficient to activate calcineurin. Treatment of lymphocytes with cytochalasin D, which blocks actin polymerization, inhibited cap formation and produced defects in signaling and IL-2 transcriptional induction in response to antigen-receptor signaling that were nearly identical to those seen in vav-/- cells. In transfection studies, either constitutively active Vav or Rac could complement constitutively active calcineurin to activate NF-AT-dependent transcription. CONCLUSIONS: These results indicate that Vav is required for cap formation in lymphocytes. Furthermore, the correlation between cap formation, IL-2 production and proliferation supports the hypothesis that an actin-dependent pathway is a source of specialized growth regulatory signals.

Actins↗
PubMed1998

Calcium oscillations increase the efficiency and specificity of gene expression.

Cytosolic calcium ([Ca2+]i) oscillations are a nearly universal mode of signalling in excitable and non-excitable cells. Although Ca2+ is known to mediate a diverse array of cell functions, it is not known whether oscillations contribute to the efficiency or specificity of signalling or are merely an inevitable consequence of the feedback control of [Ca2+]i. We have developed a Ca2+ clamp technique to investigate the roles of oscillation amplitude and frequency in regulating gene expression driven by the proinflammatory transcription factors NF-AT, Oct/OAP and NF-kappaB. Here we report that oscillations reduce the effective Ca2+ threshold for activating transcription factors, thereby increasing signal detection at low levels of stimulation. In addition, specificity is encoded by the oscillation frequency: rapid oscillations stimulate all three transcription factors, whereas infrequent oscillations activate only NF-kappaB. The genes encoding the cytokines interleukin (IL)-2 and IL-8 are also frequency-sensitive in a way that reflects their degree of dependence on NF-AT versus NF-kappaB. Our results provide direct evidence that [Ca2+]i oscillations increase both the efficacy and the information content of Ca2+ signals that lead to gene expression and cell differentiation.

Calcium↗