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D Rhodes

Publications and source records attributed to D Rhodes.

At least 127 records · Page 7Linked to original sources
PubMed1987

Solute Accumulation in Tobacco Cells Adapted to NaCl.

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na(+) and Cl(-) as principal solutes for this adjustment. Although the intracellular concentrations of Na(+) and Cl(-) correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na(+) and Cl(-) did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na(+) and Cl(-). The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na(+) and Cl(-) are principal components of osmotic adjustment, organic solutes also may make significant contributions.

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PubMed1987

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl.

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (+/-) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K(2)SO(4), Na(2)SO(4), sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Psipi) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Psipi differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Psipi difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na(+), K(+), Cl(-), free amino acids, or organic acids could not account for the Psipi differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na(+), K(+), and Cl(-) was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Psipi. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.

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PubMed1986

Mapping of the sites of protection on a 5 S RNA gene by the Xenopus transcription factor IIIA. A model for the interaction.

The contact points of transcription factor IIIA with the internal control region of the 5 S RNA gene of Xenopus have been investigated by probing the accessibility of the DNA in the protein-DNA complex to dimethylsulphate and to micrococcal nuclease. The results of quantitative measurements, combined with those from earlier DNase I and DNase II protection studies, are consistent with a series of multiple contacts about five base-pairs apart, or half a double-helical turn, along the whole length of the internal control region. The nine patches of contact we have mapped could correspond to nine DNA-binding fingers in the protein. A model for the overall geometry of the interaction is presented in which the protein lies on one face of the DNA double helix.

Animals↗
PubMed1986

An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA.

Transcription factor IIIA, which binds to the internal control region of the Xenopus 5S RNA gene has a novel structure consisting of nine tandemly repeated structural units. It was proposed by us that each unit interacts with about 5 bp of DNA. We show here that there is a periodicity on this scale in the DNA sequence and, by fine scale probing with nucleases, a corresponding structural repeat. Similar sequence periodicities are found in the internal control regions of other genes transcribed by RNA polymerase III, and also in the SV40 promoter and a monkey gene region to which the transcription factor Sp1 binds. We propose that transcription factor IIIA is the type of a novel class of transcription factors offering combinatorial possibilities for the specific recognition of DNA.

Animals↗
PubMed1986

Synthesis of [N]glutamate from [N]h(4) and [N]glycine by mitochondria isolated from pea and corn shoots.

Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [(15)N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [(15)N] H(4) (+) or [(15)N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [(15)N]H(4) (+) was provided, mitochondria isolated from light-grown pea shoots synthesized [(15)N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [(15)N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [(15)N]glycine was used, pea mitochondria synthesized [(15)N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [(15)N]glycine and synthesis of [(15)N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [(15)N]H(4) (+) by the [(14)N]H(4) (+) pool in the mitochondria. The majority of the [(15)N]H(4) (+) released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [(15)N]glycine and little synthesis of [(15)N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [(15)N]glutatmate was synthesized at a rate of 7% of that of [(15)N]serine synthesis by corn mitochondria.

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PubMed1986

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine.

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [(15)N]H(4) (+) prior to supplying the inhibitor. Analyses of the (15)N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [(15)N]H(4) (+), are (14)N enriched and are not apparently derived from (15)N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.

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PubMed1986

Metabolic changes associated with adaptation of plant cells to water stress.

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from (15)N labeling kinetics after substituting [(15)N]ammonium and [(15)N]nitrate for the (14)N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and gamma-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and gamma-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and gamma-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low (15)N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.

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PubMed1985

Structural analysis of a triple complex between the histone octamer, a Xenopus gene for 5S RNA and transcription factor IIIA.

This paper reports three experiments concerning the structural relationship between the Xenopus transcription factor IIIA (TFIIIA), the histone octamer and the Xenopus somatic gene for 5S RNA. Quantitative footprinting methods have been used in order to discover where and how TFIIIA and the histone octamer bind to the same gene independently and also in a triple complex. First, DNaseI and DNaseII protection experiments show that TFIIIA binds to positions 45-97 within the gene, in agreement with other workers. Second, the histone octamer takes up a unique, well-defined position with respect to DNA sequence. The nucleosome core extends to position 78 of the gene and therefore overlaps the TFIIIA binding region by approximately 35 bp. Third, it is shown that a triple complex can be formed between TFIIIA, the histone octamer and the 5S RNA gene. TFIIIA displaces the DNA from the histone surface in the 35-bp region of overlap. This has led to a three-dimensional model which explains how RNA polymerase III could interact simultaneously with transcription factors bound at the internal control region of the 5S RNA gene and the start point of transcription. The model also explains how histone H1 could repress transcription of 5S RNA genes.

Animals↗
PubMed1985

Betaine synthesis in chenopods: Localization in chloroplasts.

PLANTS FROM SEVERAL FAMILIES (CHENOPODIACEAE, GRAMINEAE, COMPOSITAE) ACCUMULATE BETAINE (GLYCINE BETAINE) IN RESPONSE TO SALT OR WATER STRESS VIA THE PATHWAY: choline --> betainal (betaine aldehyde) --> betaine. Betaine accumulation is probably a metabolic adaptation to stress. Intact protoplasts from leaves of spinach (Spinacia oleracea) oxidized [(14)C]choline to betainal and betaine, as did protoplast lysates. Upon differential centrifugation, the [(14)C]choline-oxidizing activity of lysates sedimented with chloroplasts. Chloroplasts purified from protoplast lysates by a Percoll cushion procedure retained strong [(14)C]choline-oxidizing activity (1-3 nmol/mg of chlorophyll per hr), although the proportion of the intermediate, [(14)C]betainal, in the reaction products was usually higher than for protoplasts. Isolated chloroplasts also readily oxidized [(14)C]betainal to betaine (20-100 nmol/mg of chlorophyll per hr). Light increased the oxidation of both [(14)C]choline and [(14)C]betainal by isolated chloroplasts approximately 3-fold; this light-stimulation was abolished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Similar results were obtained with another chenopod (Beta vulgaris) but not with pea (Pisum sativum), a species that accumulates no betaine. The chloroplast site for betaine synthesis in chenopods contrasts with the mitochondrial site in mammals.

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PubMed1984

l-Canavanine Transport and Utilization in Developing Jack Bean, Canavalia ensiformis (L.) DC. [Leguminosae].

l-Canavanine, the guanidinooxy structural analog of l-arginine, is an important nonprotein amino acid of many leguminous plants with nitrogen storage a major proported role. l-[Guanidinooxy-(14)C]canavanine, [(14)C] urea, and [(15)N]urea were injected separately into the fleshy, green cotyledons of 9-day old jack bean plants, Canavalia ensiformis (L.) DC. [Leguminosae]. There was significant transport of canavanine from the cotyledons to the aboveground portions of the plant, but not to the roots. Within 1.5 hours of isotope administration, the remaining labeled canavanine was divided equally between the cotyledons and the aboveground portions of the plant. During the 48-hour postinjection period, the contribution of l-[guanidinooxy-(14)C]canavanine to the total (14)carbon of the cotyledons decreased rapidly while it increased in the aboveground portions of the plant.[(14)C]Urea is degraded very rapidly; only 4.4% of the initial dose remained after 1.5 hours. Urea is catabolized so effectively within the cotyledons that not even 2% of the administered urea can be detected in tissues outside of these storage organs. [(15)N]Urea supplied to the developing coytledons leads to rapid (15)N incorporation into the amino nitrogen of glutamic acid and/or glutamine (28% (15)N abundance after 3 hours). Other amino acids are labeled but less heavily. The data are consistent with the proported role for l-canavanine of nitrogen storage within the developing cotyledons and cotyledonary canavanine is transported very effectively to the aboveground portions of the plant. It is not yet clear how efficiently this transported canavanine supports the nitrogen metabolism of the developing plant.

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PubMed1983

Location of the primary sites of micrococcal nuclease cleavage on the nucleosome core.

The positions and relative frequencies of the primary cleavages made by micrococcal nuclease on the DNA of nucleosome core particles have been found by fractionating the double-stranded products of digestion and examining their single-stranded compositions. This approach overcomes the problems caused by secondary events such as the exonucleolytic and pseudo-double-stranded actions of the nuclease and, combined with the use of high resolution gel electrophoresis, enables the cutting site positions to be determined with a higher precision than has been achieved hitherto. The micrococcal nuclease primary cleavage sites lie close (on average, within 0.5 nucleotide) to those previously determined by Lutter (1981) for the nucleases DNase I and DNase II. These similarities show that the accessible regions are the same for all three nucleases, the cleavage sites being dictated by the structure of the nucleosome core. The differences in the final products of the digestion are explained in terms of secondary cleavage events of micrococcal nuclease. While the strongly protected regions of the nucleosome core DNA are common to all three nucleases, there are differences in the relative degrees of cutting at the more exposed sites characteristic of the particular enzyme. In particular, micrococcal nuclease shows a marked polarity in the 3'-5' direction in the cutting rates as plotted along a single strand of the nucleosomal DNA. This is explained in terms of the three-dimensional structure of the nucleosome where, in any accessible region of the double helix, the innermost strand is shielded by the outermost strand on the one side and the histone core on the other. The final part of the paper is concerned with the preference of micrococcal nuclease to cleave at (A,T) sequences in chromatin.

Animals↗
PubMed1983

Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in hepatocytes by vasopressin.

Hepatocyte phosphatidylinositol 4,5-bisphosphate (4,5-P2), phosphatidylinositol 4-phosphate (4-P), and phosphatidylinositol were labeled with 3H when rats were injected intraperitoneally with 200 microCi of [2-3H] myo-inositol 18 h previously. Phosphatidylinositol 4,5-P2 and phosphatidylinositol 4-P accounted for 0.84 +/- 0.06 and 7.48 +/- 0.36%, respectively, of the total [3H] myo-inositol containing phospholipids. The breakdown of phosphatidylinositol 4,5-P2 was stimulated transiently (maximum effect seen at 15 s) and in a Ca2+-dependent manner by 10(-8) M vasopressin. Phosphatidylinositol 4-P breakdown was enhanced to a smaller, but longer, extent by vasopressin, whereas no changes in phosphatidylinositol were detected up to 120 s. Subcellular fractionation studies also showed no preferential breakdown of phosphatidylinositol in plasma membranes at 5-20 min. Only doses of vasopressin (10(-8) and 10(-7) M) in excess of those producing maximum effects on phosphorylase activation and Ca2+ efflux (10(-9) M) were effective at stimulating phosphatidylinositol 4,5-P2 breakdown. It is concluded that phosphatidylinositol 4,5-P2 breakdown induced by vasopressin in rat hepatocytes is not responsible for the mobilization of Ca2+ which leads to the activation of phosphorylase. On the contrary, it is Ca2+-dependent and appears to require the occupation of more receptors than are required for Ca2+ mobilization and phosphorylase activation.

Animals↗
PubMed1983

Eukaryotic RNA polymerase II binds to nucleosome cores from transcribed genes.

Purified RNA polymerase II from calf thymus can bind to about 15% of the nucleosome core particles prepared from mouse myeloma cells, forming a discrete complex having a sedimentation coefficient of 18S. These bound nucleosome cores are heavily enriched in transcribed DNA sequences, are deficient in histones H2A and H2B, and undergo a reversible change in structure on RNA polymerase II binding.

Animals↗
PubMed1983

C Tracer Evidence for Synthesis of Choline and Betaine via Phosphoryl Base Intermediates in Salinized Sugarbeet Leaves.

Like other chenopods, sugarbeets (Beta vulgaris L. cv Great Western D-2) accumulate glycine betaine when salinized; this may be an adaptive response to stress. The pathway of betaine synthesis in leaves of salinized (150-200 millimolar NaCl) sugarbeet plants was investigated by supplying [(14)C]formate, phosphoryl[(14)C]monomethylethanolamine ([(14)C][unk] MME) or phosphoryl[(14)C]choline ([(14)C][unk] choline) to leaf discs and following (14)C incorporation into prospective intermediates. The (14)C kinetic data were used to develop a computer model of the betaine pathway.When [(14)C]formate was fed, [unk] MME, phosphoryldimethylethanolamine ([unk] DME) and [unk] choline were the most prominent methylated products at short labeling times, after which (14)C appeared in free choline and in betaine. Phosphatidylcholine labeled more slowly than [unk] choline, choline, and betaine, and behaved as a minor end product. Very little (14)C entered the free methylethanolamines. When [(14)C][unk] MME was supplied, a small amount was hydrolyzed to the free base but the major fate was conversion to [unk] DME, [unk] choline, free choline, and betaine; label also accumulated slowly in phosphatidylcholine. Label from supplied [(14)C][unk] choline entered choline and betaine rapidly, while phosphatidylcholine labeled only slowly and to a small extent.These results are consistent with the pathway [unk] MME -->[unk] DME --> [unk] choline --> choline --> --> betaine, with a minor side branch leading from [unk] choline into phosphatidylcholine. This contrasts markedly (a) with the pathway of stress-induced choline and betaine synthesis in barley, in which phosphatidylcholine apparently acts as an intermediate (Hitz, Rhodes, Hanson 1981, Plant Physiol 68: 814-822); (b) with choline biogenesis in mammalian liver and microorganisms. Computer modeling of the experimental data pointed strongly to regulation at the [unk] choline --> choline step, and also indicated that the rate of [unk] choline synthesis is subject to feedback inhibition by [unk] choline.

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PubMed1981

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves.

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [(14)C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [(14)C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [(14)C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.The following experiments, in which tracer amounts of [(14)C]ethanolamine or [(14)C]formate were supplied to wilted barley leaves, implicated phosphoryl and phosphatidyl bases as intermediates in the methylation steps between ethanolamine and phosphatidylcholine. Label from both [(14)C]ethanolamine and [(14)C]formate entered phosphorylmonomethylethanolamine and phosphorylcholine very rapidly; these phosphoryl bases were the most heavily labeled products at 15 to 30 minutes after label addition and lost label rapidly as the fed (14)C-labeled precursor was depleted. Phosphatidylmonomethylethanolamine and phosphatidylcholine were also significantly labeled from [(14)C]ethanolamine and [(14)C]formate at early times; the corresponding free bases and nucleotide bases were not. Addition of a trapping pool of phosphorylcholine reduced [(14)C]ethanolamine conversion to both phosphatidylcholine and betaine, and resulted in accumulation of label in the trap.A computer model of the synthesis of betaine via phosphatidylcholine was developed from (14)C kinetic data. The model indicates that about 20% of the total leaf phosphatidylcholine behaves as an intermediate in betaine biosynthesis and that a marked decrease (>/=2-fold) in the half-life of this metabolically active phosphatidylcholine fraction accompanies wilting. Dual labeling experiments with [(14)C]choline and [(3)H]glycerol confirmed that the half-life of the choline portion of phosphatidylcholine falls by a factor of about 2 in wilted leaves.

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