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Biomedical subjects

C R Stewart

Publications and source records attributed to C R Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1988

Cyanide-resistant respiration in light- and dark-grown soybean cotyledons.

Measurements of respiration were made on intact tissue and mitochondria isolated from soybean (Glycine max [L.] Merr. cv ;Corsoy') cotyledons from seedlings of different ages grown in light and darkness. Effects of cyanide (KCN) and salicylhydroxamic acid (SHAM) on O(2) uptake rates were determined. O(2) uptake was faster in light-grown tissue and was inhibited by both KCN and SHAM in all except light-grown tissue older than 9 days. Both inhibitors stimulated O(2) uptake in tissues more than 9 days old. Mitochondria in which O(2) uptake was coupled to ATP synthesis were isolated from all tissues. O(2) uptake by mitochondrial preparations from light- and dark-grown cotyledons was equally sensitive to KCN. Similarly, age did not affect KCN sensitivity, but sensitivity to SHAM declined with age both in the presence and absence of KCN. Estimated capacities of the cytochrome and alternative pathways of the mitochondrial preparations indicated considerably larger cytochrome than alternative pathway capacities. The cytochrome pathway capacities paralleled the state 3 mitochondrial respiration rates, which increased from day 5 to day 7 then declined thereafter. The alternative pathway capacities were not affected by light. The uncoupler, p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), increased the flow of electrons through the cytochrome pathway at the expense of flow through the alternative pathway in isolated mitochondria. However, the combined capacities did not exceed the rate in the presence of FCCP. The results are interpreted to indicate that the stimulation of respiration by KCN and SHAM observed in the 12-day-old green cotyledons and previously observed in older soybean leaves is not explained by characteristics of the mitochondria.

Journal Article↗
PubMed1988

Occurrence of alternative respiratory capacity in soybean and pea.

Capacity for the alternative respiratory pathway was assessed in leaf and root tissue of male-sterile and fertile soybean (Glycine max [L.] Merr.) plants and in leaf, embryonic axis, and epicotyl tissue as well as isolated mitochondria of pea (Pisum sativum L.) by measurement of oxygen uptake in the presence and absence of KCN and salicylhydroxamic acid. Male-sterile and fertile soybean tissues showed similar responses to the inhibitors, and both possessed a capacity for alternative respiration. We also found that tissue and isolated mitochondria from ;Progress No. 9' pea possessed alternative respiratory capacity similar to that of ;Alaska' pea.

Journal Article↗
PubMed1987

Abscisic Acid accumulation is not required for proline accumulation in wilted leaves.

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.

Journal Article↗
PubMed1986

Alternative Respiratory Path Capacity in Plant Mitochondria: Effect of Growth Temperature, the Electrochemical Gradient, and Assay pH.

Influence of growth temperature on the capacity of the mitochondrial alternative pathway of electron transport was investigated using etiolated corn (Zea mays L.) seedlings. These seedlings were grown to comparable size in either a warm (30 degrees C) or a cold (13 degrees C) temperature regime, and then their respiration rates were measured as O(2) uptake at 25 degrees C. The capacity of the alternative pathway (KCN-insensitive O(2) uptake) was found essentially to double in shoots of cold-grown seedlings. This increased capacity slowly developed over several days growth in the cold, but was lost within 1 day when the seedlings were exposed to a warm regime. When mitochondria were isolated from the shoots of these seedlings, a greater potential for flow through the alternative path was observed in mitochondria from the cold-grown seedlings with all substrates used (an average increase of 84%). Using exogenous NADH as the substrate, the effect of the electrochemical gradient on measurable capacities of the cytochrome and alternative pathways was investigated in mitochondria from both etiolated seedlings and thermogenic spadices. The uncoupler FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone) was used to diminish the electrochemical gradient when desired. In corn (Zea mays L.) shoot and mung bean (Vigna radiata L.) hypocotyl mitochondria, which have relatively low capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the electrochemical gradient was found not to influence the potential for flow through the alternative path. However, in mitochondria from skunk cabbage (Symplocarpus foetidus L.) and voodoo lily (Sauromatum guttatum Schott) spadices, which have high capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the gradient occurred at the expense of flow through the alternative pathway. These results suggest that in mitochondria of thermogenic spadices, the combined capacities of the cytochrome and alternative paths exceed the capacity of the exogenous NADH dehydrogenase. The effect of assay pH on measurable capacities of the cytochrome and alternative paths was determined over a pH range of 5.6 to 8.8 using exogenous NADH as the mitochondrial substrate. When the electrochemical gradient was present, it limited the electron transport rate and little effect of assay pH was observed. However, when formation of the gradient was prevented through inclusion of FCCP, measurable capacities of the cytochrome and alternative paths were found to be greatly influenced by pH. This experiment also revealed that the potential for respiratory control is largely dependent upon the assay pH.

Journal Article↗
PubMed1986

Effects of KCN and Salicylhydroxamic Acid on Respiration of Soybean Leaves at Different Ages.

Measurements of respiration were made on leaf discs from glasshouse-grown soybean (Glycine max [L.] Merr. cv ;Corsoy') plants in the presence and absence of cyanide (KCN) and salicylhydroxamic acid (SHAM). O(2) uptake by mature leaves measured at 25 degrees C was stimulated by 1 millimolar KCN (63%) and also by 5 millimolar azide (79%). SHAM, an inhibitor of the alternative oxidase and a selection of other enzymes, also stimulated O(2) uptake by itself at concentration of 10 millimolar. However, in combination, KCN and SHAM were inhibitory. The rate of O(2) uptake declined consistently with leaf age. The stimulation of O(2) uptake by KCN and by SHAM occurred only after a certain stage of leaf development had been reached and was more pronounced in fully expanded leaves. In young leaves, O(2) uptake was inhibited by both KCN and SHAM individually. The uncoupler, p-trifluoromethoxy carbonylcyanide phenylhydrazone, stimulated leaf respiration at all ages studied, the stimulation being more pronounced in fully expanded leaves. The uncoupled rate was inhibited by KCN and SHAM individually. The capacity of the cytochrome path declined with leaf age, paralleling the decline in total respiration. However, the capacity of the alternative path peaked at about full leaf expansion, exceeding the cytochrome capacity and remaining relatively constant. These results are consistent with the presence in soybean leaves of an alternative path capacity that seems to increase with age, and they suggest that the stimulation of O(2) uptake by KCN and NaN(3) in mature leaves was mainly by the SHAM-sensitive alternative path. The stimulation of O(2) uptake by SHAM was not expected, and the reason for it is not clear.

Journal Article↗
PubMed1986

The effects of benzyladenine, cycloheximide, and cordycepin on wilting-induced abscisic Acid and proline accumulations and abscisic Acid- and salt-induced proline accumulation in barley leaves.

Benzyladenine inhibits proline accumulation in wilted, abscisic acid (ABA)-treated, and salt-shocked barley leaves. It does not affect ABA accumulation or disappearance in wilted leaves. Inhibition of proline accumulation in salt-shocked leaves was observed both when benzyladenine was added at the beginning of or after salt treatment. Cycloheximide (CHX) and cordycepin inhibited both ABA and proline accumulations in wilted barley leaves and proline accumulation in ABA-treated leaves. In salt-shocked leaves, cordycepin inhibited proline accumulation when added after salt treatment but before proline began to accumulate but not when added after the onset of proline accumulation. CHX delayed the accumulation of proline in salt-shocked leaves but, after a period of time, proline accumulated in the CHX-treated leaves at rates comparable to the salt-treated control. This delay and subsequent accumulation was observed when CHX was added before, during, and after salt treatment. However, the earlier in the salt treatment period that CHX was given, the longer was the observed delay. These results are interpreted to indicate that gene activation is involved in proline accumulation in response to wilting, to ABA, and to salt in barley leaves. This gene activation is in addition to the gene activation that is required for ABA accumulation in wilted leaves. If ABA accumulation is required for proline accumulation in wilted barley leaves, then two sets of gene activation are involved in wilting-induced proline accumulation. All of our results are consistent with this possibility but do not prove it. The inhibition of proline accumulation by benzyladenine is probably neither due to an effect on gene activation nor to an effect on the ABA level.

Journal Article↗
PubMed1985

Cloning and mapping of the SPO1 genome.

Many of the XbaI, EcoRI, KpnI, and BglII fragments of bacteriophage SPO1, accounting for about 65% of the genomic sequences, were cloned in Bacillus subtilis. Four of the EcoRI fragments were specifically refractory to cloning in both Escherichia coli and B. subtilis, probably because of expression of deleterious genes carried on the SPO1 fragments. To permit complete identification of the regions cloned, the SPO1 restriction map has been extended to include the XbaI fragments and the previously unmapped KpnI fragments. Markers for 26 of the 39 known genes have been located on specific cloned fragments, permitting more precise determination of the positions of most of the genes. One cloned SPO1 fragment was inhibitory to SPO1 development.

Bacillus subtilis↗
PubMed1985

Transcription of Bacillis subtilis plasmid pBD64 and expression of bacteriophage SPO1 genes cloned therein.

Plasmid pBD64, a vector which is useful for cloning in Bacillis subtilis (T. J. Gryczan, A. G. Shivakumar, and D. Dubnau (1980), J. Bacteriol. 141, 246-253), has at least three substantial transcription units. Two of these include the single EcoRI, XbaI, and BamHI sites, while the other includes the single BglII site. Each of these transcripts was synthesized in the counterclockwise direction, relative to the pBD64 restriction map. No transcripts were detected in the opposite direction. Infection by bacteriophage SPO1 caused a substantial decrease in each of these transcripts. No new pBD64 transcripts were detected during SPO1 infection. Various SPO1 genes, cloned at several of these pBD64 sites, were tested for expression by observing their capacity to complement SPO1 mutants. Several middle and late genes were expressed substantially, regardless of the orientation in which the fragments were inserted. Since transcription from the vector could cause expression only in one orientation, this argues that the necessary transcription originated at SPO1 promoters, and, thus, that SPO1 middle and late promoters can be active in thymine-containing DNA.

Bacillus subtilis↗
PubMed1985

Relationship between Stress-Induced ABA and Proline Accumulations and ABA-Induced Proline Accumulation in Excised Barley Leaves.

When excised second leaves from 2-week-old barley (Hordeum vulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 0.3 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABA-treated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation.

Journal Article↗
PubMed1984

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism.

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0(2) uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited big up tri, open(1)-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.The effect of T4C on proline metabolism in detached green barley leaves was investigated. T4C inhibited proline oxidation in turgid leaves, increasing the proline content of these leaves slightly. In wilted leaves (that are synthesizing proline rapidly), T4C inhibited proline synthesis, which resulted in a decrease in the proline content of the leaves. big up tri, open(1)-pyrrolidine-5-carboxylic acid reductase (the last enzyme in proline synthesis) was not inhibited by T4C, and thus T4C's influence is prior to that step of the synthetic pathway. T4C had no influence on the incorporation of proline into protein.

Journal Article↗
PubMed1984

Energetics of proline transport in corn mitochondria.

The mechanism of proline entry into the matrix region of isolated corn mitochondria (Zea mays L. Mo17 x B73) was investigated by measuring osmotically induced changes of mitochondrial size (changes in A(520)) in combination with oxygen uptake measurements. Using NADH oxidation to generate the electrochemical gradient, we have determined that proline transport is stereospecific and that it can be inhibited by the proline analog l-thiazolidine-4-carboxylic acid.The energetics of proline transport was investigated by measuring the effects of FCCP (p-trifluoromethoxycarbonyl cyanide phenylhydrazone) and valinomycin on mitochondrial swelling and substrate oxidation. Proline transport and resulting oxidation were found to be partially dependent upon the energy of the electrochemical gradient. At low proline concentrations, entry was found to be primarily independent of the gradient (based on insensitivity to FCCP), whereas at higher proline concentrations a gradient-dependent mechanism became involved. Results with valinomycin indicated that proline transport and oxidation are dependent upon the pH potential across the membrane rather than the electrical (membrane) potential.

Journal Article↗
PubMed1984

Steady state proline levels in salt-shocked barley leaves.

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na(+), and K(+) were measured at intervals. Na(+) and K(+) concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na(+) concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na(+) concentration. For a given Na(+) concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

Journal Article↗
PubMed1984

Dissection of HA20, a double mutant of bacteriophage SPO1.

HA20, a mutant of Bacillus subtilis phage SPO1, is deficient in both DNA replication and late transcription. HA20 contains mutations in two different genes, which suggested that the two effects might be caused independently. However, single-mutation derivatives, affected only in gene 27, were deficient for both activities. Thus, a single mutation apparently affects both DNA replication and late transcription.

Bacteriophages↗
PubMed1983

Effects of NaCl on Proline Synthesis and Utilization in Excised Barley Leaves.

Proline accumulation in NaCl-treated excised barley (Hordeum vulgare var Larker) leaves was studied. Leaves were treated by placing the cut end in NaCl solutions and allowing the salt to enter the leaf via the transpiration stream. Leaves treated this way maintained turgor while the sodium content increased and the osmotic potential decreased. Proline began accumulating after 12 hours and continued accumulating over the subsequent 12-hour period at an average rate of 0.6 micromoles per hour per gram fresh weight.During the time proline was accumulating, [(14)C]glutamate was added to measure the effects of salt on proline synthesis from glutamate and [(14)C] proline was added in separate experiments to determine the effect of salt on proline utilization. Salt treatment dramatically increased proline synthesis from glutamate. Proline utilization by oxidation and for protein synthesis was decreased by 50 and 60%, respectively, by the salt treatment.These effects are similar to the effects of drought and abscisic acid in barley leaves. The results indicate that common mechanisms cause proline to accumulate under these different stresses.

Journal Article↗
PubMed1983

Photorespiratory glycine metabolism in corn leaf discs.

The total glycine pool in Zea mays L. Mo17xB73 leaf discs was measured after steady state photosynthesis in 50%, 21% and 1% O(2). The glycine pool was a function of O(2) concentration; it was largest in 50% O(2) and smallest in 1% O(2). Incubation of discs with methyl hydroxybutynoic acid in 21% O(2) in the light caused an accumulation of carbon in glycolate. This accumulation was O(2) sensitive, as subsequent photosynthetic periods in 50%, 21%, and 1% O(2) resulted in the largest glycolate pool in 50% O(2) and the smallest in 1% O(2). At the same time, the O(2)-dependent increase in the glycine pool was eliminated. After untreated leaf discs reached steady state photosynthesis in 21% O(2), measurements made subsequently in darkness, or in 1% O(2) in the light, showed that the glycine pool decreased. On the basis of these results, we conclude that a major portion of the total glycine pool in corn is an intermediate in the photorespiratory glycolate pathway. Considering both the rate of decay of the glycine pool in the dark and the rate of decay of the glycine pool after changing from 21% to 1% O(2), we conclude that this glycine pool is turning over slowly.

Journal Article↗
PubMed1982

Proline Oxidation in Corn Mitochondria : Involvement of NAD, Relationship to Ornithine Metabolism, and Sidedness on the Inner Membrane.

Proline-dependent oxygen uptake in corn mitochondria (Zea mays L. B73 x Mo17 or Mo17 x B73) occurs through a proline dehydrogenase (pH optimum around 7.2) bound to the matrix side of the inner mitochondrial membrane. Sidedness was established by determining the sensitivity of substrate-dependent ferricyanide reduction to antimycin and FCCP (P-trifluoromethoxycarbonylcyanide phenylhydrazone). Proline dehydrogenase activity did not involve nicotinamide adenine dinucleotide reduction, and thus electrons and protons from proline enter the respiratory chain directly. Delta(1)-Pyrroline-5-carboxylate (P5C) derived from proline was oxidized by a P5C dehydrogenase (pH optimum approximately 6.4). This enzyme was found to be similar to proline dehydrogenase in that it was bound to the matrix side of the inner membrane and fed electrons and protons directly into the respiratory chain.Ornithine-dependent oxygen uptake was measurable in corn mitochondria and resulted from an ornithine transaminase coupled with a P5C dehydrogenase. These enzymes existed as a complex bound to the matrix side of the inner membrane. P5C formed by ornithine transaminase was utilized directly by the associated P5C dehydrogenase and was not released into solution. Activity of this dehydrogenase involved the reduction of nicotinamide adenine dinucleotide.

Journal Article↗