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D W Sutton

Publications and source records attributed to D W Sutton.

15 recordsLinked to original sources
PubMed1992

Synthetic cryIIIA gene from Bacillus thuringiensis improved for high expression in plants.

A 1974 bp synthetic gene was constructed from chemically synthesized oligonucleotides in order to improve transgenic protein expression of the cryIIIA gene from Bacillus thuringiensis var. tenebrionis in transgenic tobacco. The crystal toxin genes (cry) from B. thuringiensis are difficult to express in plants even when under the control of efficient plant regulatory sequences. We identified and eliminated five classes of sequence found throughout the cryIIIA gene that mimic eukaryotic processing signals and which may be responsible for the low levels of transcription and translation. Furthermore, the GC content of the gene was raised from 36% to 49% and the codon usage was changed to be more plant-like. When the synthetic gene was placed behind the cauliflower mosaic virus 35S promoter and the alfalfa mosaic virus translational enhancer, up to 0.6% of the total protein in transgenic tobacco plants was cryIIIA as measured from immunoblot analysis. Bioassay data using potato beetle larvae confirmed this estimate.

Animals↗
PubMed1992

Elevation of organ resistance due to leukocyte perfusion.

Leukocytes are larger and considerably less deformable than erythrocytes, thereby causing a disproportionate effect on local blood flow, which despite their low concentration, may be significant at the organ level. To investigate the degree to which circulating leukocytes affect whole organ resistance, a hemodynamically isolated rat gracilis muscle was perfused in situ under well-controlled conditions. Comparison of organ pressure-flow data from vasodilated vasculature using normal physiological cell concentrations and perfusion pressures indicates that leukocytes (60-75% neutrophils) provide approximately 22% of the whole blood resistance despite their relatively small cell volume fraction of approximately 0.1%. On a single cell basis, each leukocyte imposes a resistance elevation equivalent to that of approximately 750 erythrocytes. Furthermore, when leukocytes are activated via pretreatment using N-formyl-methionyl-leucyl-phenylalanine or endotoxin, they show a higher resistance, accounting for 50-60% of the total resistance. These findings indicate that leukocytes play a significant role in normal skeletal muscle organ perfusion and may be a major determinant of organ perfusion during disease states.

Animals↗
PubMed1991

The pressure-flow relation for plasma in whole organ skeletal muscle and its experimental verification.

The whole-organ pressure-flow relation in resting rat skeletal muscle is examined for the flow of plasma. Due to the small size of the blood vessels in this organ, inertia and convective forces in the blood are negligible and viscous forces dominate. Direct measurements in the past have shown that skeletal muscle blood vessels are distensible. Theoretical formulations based on these measurements lead to a third order polynomial model for the pressure-flow relation. The purpose of the current study is to examine this relation experimentally in an isolated muscle organ. A high precision feedback controlled pump is used to perfuse artificial plasma into the vasodilated rat gracilis muscle. The results indicate that the pressure-flow curve in this tissue is nonlinear in the low flow region and almost linear at physiological flow rates, following closely the third order polynomial function. Vessel fixation with glutaraldehyde causes the curves to become linear at all pressures, indicating that vessel distention is the primary mechanism causing the nonlinearity. Furthermore, the resistance of the post-fixed tissue is determined by the pressure at which the fixative is perfused. At fixation pressures below 10 mmHg, the resistance is three times higher than in vessels fixed at normal physiological pressures. Dextran (229,000 Dalton) is used to obtain Newtonian perfusates at different viscosities. The pressure-flow relation is found to be linearly dependent on viscosity for all flow rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A high precision dual feedback pump for unsteady perfusion of small organs.

A dynamic pump system is described for perfusion of small organs with whole blood. The pump system was designed with the following aims: Very low flowrates to perfuse single organs in small rodents; high dynamic response for pressure or flow to permit experimenting with a harmonic signal at frequencies up to 20 Hz or by way of sharp step transients in less than 10 msec; high precision to allow detection of fine physiological details, and minimum blood cell trauma or cell activation by use of a piston principle. Representative pressure-flow curves are shown for the rat gracilis muscle after vasodilation. The curves are highly reproducible and serve as a complimentary dataset for microvascular observations in the same organ.

Animals↗
PubMed1989

Hemodynamics at low flow in resting vasodilated rat skeletal muscle.

The low flow arterial pressure-flow relationship and zero-flow pressure (ZFP) are investigated in the hemodynamically isolated gracilis muscle using a high precision pump. The muscle is kept in situ with dilated vasculature. During steady-state perfusion, using a plasma-like medium, the pressure-flow curve is nonlinear with positive arterial ZFP of 3.5-12 mm Hg when normal central circulation pressure is present. When the central circulation is stopped the ZFP reduces to zero. Addition of nonaggregated red blood cells (RBCs) results in no significant increase in the ZFP; however, introduction of aggregated RBCs (with dextran, 77 kDa) causes a 9.4 +/- 1.2 mmHg elevation. The positive ZFPs observed using plasma-like and dispersed RBC perfusions are found to be caused by a back pressure from the central circulation via collateral arterioles. A single-step reduction of the arterial flow rate from a finite value to zero results in a ZFP, which decreases for more than a minute before steady state is reached. During harmonic flow inputs with oscillations down to zero flow, an increase in the ZFP is detected near 0.09 Hz and continues to rise up to the test limit of 10 Hz. Our results suggests that in vasodilated skeletal muscle three independent mechanisms exist, collateral flow, cell aggregation, and unsteady perfusion, which may cause a positive arterial ZFP.

Animals↗
PubMed1988

Cell separation in the buffy coat.

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.

Blood Platelets↗
PubMed1979

Purification and characterization of the crown gall specific enzyme nopaline synthase.

Nopaline synthase of sunflower (Helianthus annuus L.) crown gall tissue induced by Agrobacterium tumefaciens strain C58 or T37 (nopaline utilizers) was purified to homogeneity as judged by analytical disc gel electrophoresis. The native enzyme elutes from a column of Ultrogen AcA 34 as a single peak with an estimated molecular weight of 158,000. The dissociated enzyme migrates on NaDodSO4-polyacrylamide gels as a single band with a molecular weight of 40,000. Thus, the native enzyme appears to be composed of four equal-weight subunits. Nopaline synthesizing activity is found exclusively in crown gall tissues induced by strains of A. tumefaciens that utilize nopaline (e.g., C58 and T37). We found the same tissue specificity for the purified protein that we believe represents nopaline synthase. The results of kinetic studies of the purified enzyme are consistent with a ter-bi rapid-equilibrium random-order mechanism. Nopaline synthase is probably responsible for the in vivo synthesis of both N2-(1,3-dicarboxypropyl)arginine (nopaline) and N2-(1,3-dicarboxypropyl)ornithine (ornaline) in crown gall tissues since substrate specificities and Km values do not change during purification.

Amino Acid Oxidoreductases↗
PubMed1979

Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines.

Total RNA isolated from three octopine-type crown gall lines contains sequences homologous to specific regions of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens strain 15955. A comparison of transcripts in these three tumor lines suggests that tumor cells transcribe various sequences within a sector of plasmid DNA of 13 x 10(6) daltons and that transcription may not be uniform across the plasmid derived sequences (T-DNA). Transcription of T-DNA by octopine-type tumors occurs at four major sites. The levels of transcription occurring at three of these sites appear to vary considerably among the three tumor lines investigated. Part of this variability may reflect differences in the organization and copy number of T-DNA. One of the transcription sites maps within a region of DNA with common sequence homology with all Ti plasmids. Varying amounts of transcript homologous to this region of T-DNA are present in all three tumor lines. It is suggested that transcription of these conserved sequences in the plant may have significance regarding the mechanism of tumorigenesis.

Arginine↗
PubMed1978

Characterization of the enzyme responsible for nopaline and ornaline synthesis in sunflower crown gall tissues.

Extracts prepared from sunflower (Helianthus annuus L.) crown gall tissues induced by Agrobacterium tumefaciens strains C58 and T37 (nopaline utilizers) catalyze the synthesis of nopaline and ornaline. These compounds are not synthesized in extracts of crown gall tissues induced by strains B6, 15955 (octopine utilizers), and AT1 (utilizes neither octopine nor nopaline) or in extracts of habituated sunflower callus. Both synthetic activities require NADPH, alpha-ketoglutarate, and either arginine or ornithine; histidine and lysine will not substitute. Incorporation of arginine or ornithine into product is inhibited by the other substrate but not by histidine or lysine. On the basis of inhibition and K(m) data, both activities appear to be catalyzed by one enzyme and the same enzyme is apparently present in crown gall tissues induced by strains C58 and T37.

Journal Article↗
PubMed1976

Calculation of absolute rates of RNA synthesis, accumulation, and degradation in tobacco callus in vivo.

Uptake and incorporation data were used to calculate an absolute rate of ribonucleic acid (RNA) synthesis by assuming that incorporation of radioactive nucleosides into RNA was a first-order function of the nucleoside triphosphate cellular pool. Homogenized tissue was separated into an RNA and cellular pool fraction. Methods were developed to directly measure the specific radioactivity of specific nucleosides in each fraction. Exogenous [3H]uridine was taken up by the cells, converted to I[3H]uridine triphosphate ([3H]UTP) and [3H]cytidine triphosphate ([3H]CTP), and incorporated into RNA. All of the radioactivity in RNA was contained in either uridine or cytidine. The average rate of RNA synthesis (20 mg of RNA (g of RNA)-1h-1) was calculated from changes in the specific radioactivity of uridine or cytidine in RNA and UTP or CTP in the pool. Since ribosomal RNA does not turn over in exponentially growing tissue, its rate of synthesis was measured (14 mg g-1h-1) and compared with the rate of accumulation of RNA (12 mg g-1h-1). The similarity between ribosomal RNA synthesis and total RNA accumulation confirmed the assumption that these methods measured an absolute rate of RNA synthesis.

Cytosine Nucleotides↗
PubMed1976

A chemical and physical method for determining the complete base composition of plant DNA.

Two physical methods are routinely used to determine the base composition of DNA. One measures the temperature corresponding to the midpoint of the absorbance rise (TM) and relates it to base composition with the equation, TM = 41 (dG + dC) + 69, the other measures buoyant density (rho) and relates it to base composition rho = 0.098(dG + dC) + 1.6535. The base composition of DNA from various sources was first determined by a chemical method and these values compared to those determined by the physical methods. Higher plants contained up to 7 mol% 5-methyldeoxycytidine in their DNA and in all cases tested deoxyguanosine = deoxycytidine + 5-methyldeoxycytidine. After determining that TM was unaffected by the amount of 5-methyldeoxycytidine in DNA, the mol% of dA, dT, dG, and the total of dC plus 5-methyldeoxycytidine for any DNA could be calculated. Buoyant density on the other hand, was lowered 0.004 g . cm-3 for every 6.3 mol% 5-methyldeoxycytidine. Therefore, both physical parameters were related to the mole fraction of 5-methyldeoxycytidine by the following equation: (see article). With a value of r 5-methyldeoxycytidine an estimation of deoxycytidine was made. The resultant values agreed with the chromatographic determinations.

Deoxyribonucleosides↗
PubMed1972

Protein Metabolism in Cultured Plant Tissues: III. Changes in the Rate of Protein Synthesis, Accumulation, and Degradation in Cultured Pith Tissue.

During the transition of tobacco (Nicotiana tabacum) pith tissue to callus tissue, there were changes in the composition of the soluble amino acid pools, in the distribution of amino acids between pool and protein, and in the synthesis, accumulation, and degradation of proteins. The size of the leucine pool decreased from 90 nanomoles per gram fresh weight in fresh pith to 20 nanomoles in 24-hour cultured pith, followed by a return to 90 nmoles in pith cultured longer than 5 days. The latter value is the same as that reported for exponentially growing callus cells. Many other pool amino acids changed as dramatically. However, they always approached callus levels after 5 days of culturing. The total amino acid content of pith tissue (the sum of both pool and protein) remained unchanged during culturing. The value for total amino acid content (34 to 42 nanomoles per gram fresh weight) was also similar to that found in callus. The distribution of amino acids between pool and protein did change during culturing. The transition of pith tissue with 88% of its total amino acids free in the soluble pool to callus with 92% of its amino acids in protein was further characterized by changes in protein metabolism. Both protein synthesis and accumulation increased over the first 50 hours in culture to a maximum rate of 45 milligrams protein synthesized gram protein(-1) hour(-1). After 50 hours in culture, the rate of protein accumulation decreased to equal the rate of fresh weight accumulation (10 mg g(-1) hour(-1)). However, protein synthesis continued at a high rate for several days, suggesting protein degradation was turned on by this time. By 5 days protein synthesis had decreased to a rate similar to that of callus.

Journal Article↗
PubMedDate not supplied

The pressure-flow relation in resting rat skeletal muscle perfused with pure erythrocyte suspensions.

In spite of numerous investigations of erythrocyte rheology, there is limited information about the influence of erythrocyte suspensions on whole organ pressure-flow relationships. In this study, we present whole organ pressure-flow curves for resting vasodilated gracilis muscle of the rat, in which the microanatomy and vessel properties have been determined previously. For pure erythrocyte suspensions from donor rats, the organ resistance increases only mildly with perfusion time (less than a 5% shift over a one-hour perfusion time), while in contrast, erythrocyte suspensions containing leukocytes show an increases of resistance near 100% over a period of 25 min. Variation in pressure-flow curves in the muscle at the same arterial hematocrit between different rats is less than 15%. The pressure-flow relation for pure erythrocyte suspensions depends on hematocrit. Shear thinning is exhibited at high hematocrits, while Newtonian behavior is approached at arterial hematocrits below 15%. The whole organ apparent viscosity for pure erythrocyte suspensions (normalized by cell-free plasma resistance) is a non-linear function of hematocrit; at physiological pressures, it reaches values comparable to those of apparent viscosities measured in rotational viscometers or in in vitro tube flow (diameters greater than 0.8 mm). The apparent viscosities estimated from the whole organ experiments tend to be higher than those measured in straight tubes under in vitro conditions. The pressure-flow curves for pure erythrocyte suspensions are shifted towards lower pressures than the curves for mixed suspensions of erythrocytes at the same hematocrit and with leukocytes at physiological cell counts. These acute experiments show that pure erythrocyte suspensions yield highly reproducible resistances in the skeletal muscle microcirculation with dilated arterioles. Relative apparent viscosities measured in vivo are higher than those measured in straight glass tubes of comparable dimesions.

Animals↗