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D Rhodes

Publications and source records attributed to D Rhodes.

At least 109 records · Page 6Linked to original sources
PubMed1989

A novel method for the purification of the Xenopus transcription factor IIIA.

We have developed a quick and simple purification method that yields large quantities of the Xenopus zinc finger protein transcription factor IIIA (TFIIIA). The protein is purified in the form of the 7S storage particle (TFIIIA/5S RNA complex) found in the ovaries of immature Xenopus. Our method yields 0.5 to 1 mg of pure 7S particle per ovary. It involves a high speed centrifugation step, fractionation on a gel filtration column and a precipitation step using calcium chloride. TFIIIA purified using this protocol retains full DNA binding activity and has the expected zinc ion content.

Animals↗
PubMed1989

The DNA binding site of the Xenopus transcription factor IIIA has a non-B-form structure.

On the basis of nuclease digestion studies we proposed that the DNA binding site of transcription factor IIIA (TFIIIA) may have an overall structure with A-type rather than B-type characteristics. This proposal was substantiated by the crystal structure of a part of the TFIIIA binding site. Recently, however, it has been reported that the binding site for TFIIIA is B-form in solution, thus implying that the conformation present in crystals is not the structure in solution. We have carried out a study using comparative circular dichroism (CD) spectroscopy of a number of double stranded deoxyoligonucleotides of different sequence, and known crystal structure. The correlation we have found between CD characteristics and certain structural parameters indicates that the solution and crystal structures of the TFIIIA binding site are closely related. This structure may be classed as an intermediate type, between A-form and B-form DNA.

Animals↗
PubMed1989

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine.

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of (15)N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [(14)N]asparagine and [(14)N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-(15)N- and double-(15)N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[(15)N] abundance of these amino acids was determined. We demonstrate that this approach to (15)N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-(15)N:double-(15)N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with (15)N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [(15)N]H(4) (+) are (14)N-enriched and must be derived primarily from protein turnover.

Journal Article↗
PubMed1989

Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma.

Chenopods synthesize betaine in the chloroplast via a two-step oxidation of choline: choline --> betaine aldehyde --> betaine. Our previous experiments with intact chloroplasts, and in vivo(18)O(2) labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] Plant Physiol 88: 695-702). Here, we report the detection of such an activity in vitro. In the presence of O(2) and reduced ferredoxin, the stromal fraction from spinach (Spinacia oleracea) chloroplasts converted choline to betaine aldehyde at rates similar to those in intact chloroplasts (20-50 nanomoles per hour per milligram protein). Incorporation of (18)O from (18)O(2) by the in vitro reaction was demonstrated by fast atom bombardment mass spectrometry. Ferredoxin could be reduced either with thylakoids in the light, or with NADPH plus ferredoxin-NADP reductase in darkness; NADPH alone could not substitute for ferredoxin. No choline-oxidizing activity was detected in the stromal fraction of pea (Pisum sativum L.), a species that does not accumulate betaine. The spinach choline-oxidizing enzyme was stimulated by 10 millimolar Mg(2+), had a pH optimum close to 8, and was insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCl. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin.

Journal Article↗
PubMed1989

Development of two isogenic sweet corn hybrids differing for glycinebetaine content.

A hybrid of sweet corn, Zea mays L. (;1720'; Rogers Brothers Seed Co.), was found to be comprised of glycinebetaine-positive and glycinebetaine-deficient individuals in a 1:1 mixture. This phenomenon was traced to segregation for a single, nuclear, dominant gene determining leaf glycinebetaine content within the female inbred parent of this hybrid. Selection for homozygous recessive (glycinebetaine-deficient) and homozygous dominant (glycinebetaine-positive) genotypes of the female inbred parent enabled production of two isogenic versions of hybrid ;1720' differing with respect to a single copy of the dominant allele, by mating these female parent selections with the common homozygous recessive (glycinebetaine-deficient) male parent. These two isogenic hybrids are shown to differ by a factor of 300- to 400-fold in glycinebetaine titer of young expanding leaves of salinized plants, but exhibit no striking differences in the levels of free amino acids or the level of N-methylnicotinic acid (nicotinic acid betaine; trigonelline). The only significant difference between the two hybrids in terms of amino acid composition was found to be in the level of alanine under nonsalinized conditions. The betaine-deficient hybrid exhibited a 14% lower alanine level than the betaine-positive hybrid. Betaine deficiency was not associated with altered stress-induced accumulation of amino acids such as proline, serine, and asparagine plus aspartate, attesting to the high specificity of the genetic difference between these isogenic hybrids with respect to betaine accumulation. This germplasm offers unique opportunities to test whether a single dominant allele determining stress-induced betaine accumulation capacity influences stress resistance in maize.

Journal Article↗
PubMed1989

Genotypic Variation for Glycinebetaine among Public Inbreds of Maize.

Screening of a range of public maize (Zea mays L.) inbred lines for glycinebetaine (betaine) content over two growing seasons (1987 and 1988), using fast atom bombardment mass spectrometry, has identified 19 public inbred lines which all exhibit low betaine levels (<100 nanomoles per gram fresh weight). These include common inbreds such as A188, A619, B37, H95, N6, and Oh43. Several inbreds exhibit high betaine levels (3000 to 10000 nanomoles per gram fresh weight); in these strongly betaine-positive inbreds, betaine levels tended to be, on average, 1.38-fold greater in the 1988 growing season presumably in part due to field water deficits experienced during the drought of 1988. Where several different sources of the same inbred line were available (including cytoplasmic male sterile and restored lines of A632, B37, B73, Oh43, and WF9), betaine levels were found to be similar when the inbreds were tested in the same environment. Because W22-R/r-X1 was found to be strongly betaine-positive, it should be possible to map the putative recessive gene(s) determining betaine deficiency to specific chromosome(s) from monosomics resulting from crosses between W22-R/r-X1 and betaine-deficient lines.

Journal Article↗
PubMed1988

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture.

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.

Alcohol Dehydrogenase↗
PubMed1988

Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA.

The short sequence motif named 'zinc finger', first recognized repeated in tandem in the Xenopus transcription factor IIIA (TFIIIA), is also found in the yeast transcriptional activator SWI5 (ref. 3) and many other regulator proteins. Embedded in the 709-amino-acid polypeptide chain of SWI5 are three tandemly repeated zinc-finger motifs. Because the zinc fingers of TFIIIA are known to bind to DNA, it is probable that in the case of SWI5 these finger motifs also play an important, but not necessarily exclusive, role in the sequence-specific binding of the protein to DNA. To test this prediction we have expressed the 89-amino-acid sequence of the domain containing the three zinc fingers of SWI5 in Escherichia coli as a cleavable fusion protein, purified under denaturing conditions and folded in vitro. This experimental approach allows us to study directly both the metal requirement and DNA-binding properties of the isolated polypeptide. We find that zinc is required for specific DNA recognition and, most significantly, DNaseI protection studies show that the isolated three-fingered domain is sufficient for sequence-specific binding to DNA.

Amino Acid Sequence↗
PubMed1988

Structure and location of amiodarone in a membrane bilayer as determined by molecular mechanics and quantitative x-ray diffraction.

Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate.

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of alpha-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.

Journal Article↗
PubMed1988

Preliminary Genetic Studies of the Phenotype of Betaine Deficiency in Zea mays L.

Glycinebetaine-deficient inbreds of Zea mays do not exhibit a general deficiency of nitrogenous solutes; the total free amino acid levels of betaine-deficient lines are not significantly less than those of inbreds which exhibit >100-fold higher betaine levels. Betaine-deficient inbreds are characterized by extremely low betaine: total free amino acid ratios (<0.0015). Highly significant correlations are demonstrated between the expected mid-parent and observed betaine:amino acid ratios of 30 hybrids of known pedigree. In 12 hybrids constructed from a betaine-deficient male parent (inbred 1506), the observed betaine:amino acid ratios of the hybrids are proportional to the betaine:amino acid ratios of the female parents (r = 0.83). Two hybrids, 1146 x 1074 and 1146 x 1506, were chosen for further genetic analysis. The common female parent (1146) and inbred 1074 both exhibit betaine:amino acid ratios of 0.090, a value which is approximately 90-fold greater than the betaine:amino acid ratio of inbred 1506. Hybrid 1146 x 1074 exhibits almost exactly twice the betaine:amino acid ratio of hybrid 1146 x 1506. If inbred 1506 is homozygous recessive for a single nuclear gene responsible for the phenotype of betaine deficiency, and if inbreds 1146 and 1074 are homozygous dominant for this allele, then this twofold difference in betaine:amino acid ratio must be associated with the homozygous dominant and heterozygous conditions, respectively, for 1146 x 1074 and 1146 x 1506. Evidence is presented from both greenhouse and field evaluations of F(2) populations of these hybrids that a single nuclear recessive gene is most likely responsible for the phenotype of betaine-deficiency in inbred 1506. Approximately 25% of the F(2) segregants from 1146 x 1506 exhibited extremely low betaine:amino acid ratios (<0.0015), whereas 0% of the F(2) segregants from 1146 x 1074 exhibited this phenotype. The segregation patterns with respect to betaine:amino acid ratio suggest a 1:2:1 segregation ratio for homozygous recessive:heterozygous:homozygous dominant individuals within the 1146 x 1506-F(2) population.

Journal Article↗
PubMed1988

Oxygen-18 and deuterium labeling studies of choline oxidation by spinach and sugar beet.

Chenopods synthesize betaine by a two-step oxidation of choline: choline --> betaine aldehyde --> betaine. The pathway is chloroplastic; the first step has been shown in isolated spinach (Spinacia oleracea L.) chloroplasts to be O(2)- and light-dependent, the role of light being to provide reducing power (P Weigel, EA Weretilnyk, AD Hanson 1988 Plant Physiol 86: 54-60). Here, we report use of in vivo(18)O- and (2)H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O(2) and reductant: a desaturase or an oxygenase. Simple syntheses for (2)H(3)-choline, (2)H(3), (18)O-choline, and (2)H(3), (18)O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with (2)H(2)O and by analyzing newly synthesized betaine. About 15% of the (2)H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% (2)H(2)O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of (2)H from the C-2 position also occurred if choline was converted to betaine by a purified bacterial choline oxidase. To test for an oxygenase, the incorporation of (18)O from (18)O(2) into newly synthesized betaine was compared with that from (18)O-labeled choline, in light and darkness. Incorporation of (18)O from (18)O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the (18)O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of (18)O from (18)O(2) approached that from (18)O-choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic (16)O(2). These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase.

Journal Article↗
PubMed1988

Teaching nurses therapeutic conversation: a pilot study.

A 2-day video-based workshop developed for teaching psychiatrists was used to instruct learner psychiatric nurses in the conversational model of psychotherapy. A small evaluation was carried out to assess the effects of the teaching on the performance of these nurses in interviewing other learner nurses who role-played people with common emotional difficulties. A randomized groups design was used comparing the interview behaviour of experimental and control groups before and after the workshops, and at 3 months follow-up. Considered as a pilot the study was instructive, although only one significant result emerged. The conversational model of psychotherapy and the teaching workshops devised and used previously with psychiatrists can be used by nurses, including those still in training.

Adult↗
PubMed1987

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron.

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10(-8) molar and above, but had no inhibitory effects on growth at 10(-9) molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10(-8) molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as alpha-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of alpha-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10(-4) molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10(4) times greater than that required to inhibit growth. Tracer studies with (15)N demonstrate that chlorsulfuron inhibits the incorporation of (15)N into valine, leucine, and isoleucine. The alpha-amino-n-butyrate accumulated in the presence of chlorsulfuron and [(15)N]H(4) (+) was heavily labeled with (15)N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to alpha-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as alpha-amino-N donors.

Journal Article↗
PubMed1987

Determination of Betaines by Fast Atom Bombardment Mass Spectrometry : Identification of Glycine Betaine Deficient Genotypes of Zea mays.

A rapid, sensitive, and selective method for the determination of betaines is described and discussed. The method entails derivatizing the quaternary ammonium compounds to increase their sensitivity to detection by fast atom bombardment mass spectrometry. Sensitivity of detection increases markedly as the length of the carbon chain of the alcohol used to esterify the betaine carboxylic acid group is increased (C4 > C3 > C2 > C1 > C0). The lower limit of detection of glycine betaine as the n-propyl ester is 0.05 nanomole per microliter of glycerol. Betaine aldehyde can be readily derivatized to the di-n-butyl or di-n-propyl acetal derivatives which exhibit lower limits of detection of about 5 picomoles and 10 picomoles per microliter of glycerol, respectively. Accurate quantification of these compounds is accomplished by the use of deuterium labeled internal standards or quaternary ammonium compound homologs of distinct mass. Methods for the synthesis of these internal standards are reported. Some applications of these methods are illustrated with stable isotope tracer studies on the kinetics of metabolism of choline to betaine aldehyde and glycine betaine in spinach leaf discs, and the identification of several Zea mays genotypes which appear deficient in glycine betaine. Tracer studies with deuterium labeled betaine aldehyde suggest that the deficiency of glycine betaine in one sweet corn hybrid is probably not due to a deficiency in the capacity to oxidize betaine aldehyde.

Journal Article↗