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Biomedical subjects

M A Lawton

Publications and source records attributed to M A Lawton.

At least 19 recordsLinked to original sources
PubMed1995

Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis.

Benzoic acid 2-hydroxylase (BA2H) catalyzes the biosynthesis of salicylic acid from benzoic acid. The enzyme has been partially purified and characterized as a soluble protein of 160 kDa. High-efficiency in vivo labeling of salicylic acid with 18O2 suggested that BA2H is an oxygenase that specifically hydroxylates the ortho position of benzoic acid. The enzyme was strongly induced by either tobacco mosaic virus inoculation or benzoic acid infiltration of tobacco leaves and it was inhibited by CO and other inhibitors of cytochrome P450 hydroxylases. The BA2H activity was immunodepleted by antibodies raised against SU2, a soluble cytochrome P450 from Streptomyces griseolus. The anti-SU2 antibodies immunoprecipitated a radiolabeled polypeptide of around 160 kDa from the soluble protein extracts of L-[35S]-methionine-fed tobacco leaves. Purified BA2H showed CO-difference spectra with a maximum at 457 nm. These data suggest that BA2H belongs to a novel class of soluble, high molecular weight cytochrome P450 enzymes.

Antibodies↗
PubMed1995

Cloning and properties of a rice gene encoding phenylalanine ammonia-lyase.

Phenylalanine ammonia-lyase (PAL) genomic sequences were isolated from a rice (Oryza sativa L.) genomic library using a PCR-amplified rice PAL DNA fragment as a probe. There is a small family of PAL genes in the rice genome. The nucleotide sequence of one PAL gene, ZB8, was determined. The ZB8 gene is 4660 bp in length and consists of two exons and one intron. It encodes a polypeptide of 710 amino acids. The transcription start site was 137 bp upstream from the translation initiation site. Rice PAL transcripts accumulated to a high level in stems, with lower levels in roots and leaves. Wounding of leaf tissues induced ZB8 PAL transcripts to a high level. In rice suspension-cultured cells treated with fungal cell wall elicitors, the ZB8 PAL transcript increased within 30 min and reached maximum levels in 1-2 h. The transcription of the ZB8 gene was investigated by fusing its promoter to the reporter gene beta-glucuronidase (GUS) and transforming the construct into rice and tobacco plants, as well as rice suspension-cultured cells. High levels of GUS activity were observed in stems, moderate levels in roots and low levels in leaves of transgenic rice and tobacco plants. Histochemical analysis indicated that in transgenic rice the promoter was active in root apical tips, lateral root initiation sites, and vascular and epidermal tissues of stems and roots. In rice flowers, high GUS activity was observed in floral shoots, receptacles, anthers and filaments, occasionally GUS activity was also detected in lemma and awn tissues. In tobacco flowers, high GUS activity was detected in the pink part of petals. Consistent with the activity of endogenous PAL transcripts, wounding of rice and tobacco leaf tissues induced GUS activity from low basal levels. Tobacco mosaic virus (TMV) infection of tobacco leaves induced GUS activity to a high level. Fungal cell wall elicitors strongly induced GUS activity and GUS transcripts to high levels in transgenic rice suspension-cultured cells. We demonstrated that the promoter of ZB8 gene is both developmentally regulated and stress-inducible.

Amino Acid Sequence↗
PubMed1995

Direct in vivo visualization of right cardiac anatomy by fiberoptic endoscopy. Hemodynamic effects and image validation.

The authors tested the ability of a balloon-tipped fiberoptic endoscope to accurately visualize and identify right-heart anatomy in 7 anesthetized dogs. A 3.6-mm-diameter fiberoptic endoscope with a latex balloon covering the distal tip was inserted into the right atrium, where the balloon was inflated with air in 5 mL increments. Heart rate did not show changes. Mean arterial pressure and cardiac output started to show significant decreases with a balloon volume at 25 and 20 mL, respectively (n = 7). Visual image quality was excellent with a balloon volume of 10 mL or greater. With a balloon volume of 7-10 mL, the visual field was 15-20 mm in diameter. Right-heart anatomy including the right free wall, ostium of the coronary sinus, atrioventricular node area, tricuspid valve, right ventricular structures, and pulmonary arteries was clearly and accurately identified. Additionally, spatial relationships among these structures could be established. Furthermore, there was an excellent concordance between endoscopically observed images and postmortem findings. In conclusion, balloon-tipped fiberoptic endoscopy can accurately visualize normal intracardiac structures with no or minimal hemodynamic compromise.

Animals↗
PubMed1994

atpk1, a novel ribosomal protein kinase gene from Arabidopsis. I. Isolation, characterization, and expression.

Two protein kinase genes (atpk1 and atpk2) were isolated from Arabidopsis thaliana genomic DNA with a probe generated by polymerase chain reaction (PCR) using oligonucleotide primers encoding conserved eukaryotic protein kinase sequences. atpk1 and atpk2 are organized in a head-to-tail tandem array on chromosome 3 and have about 80% nucleotide sequence identity. atpk1 encodes a hydrophilic polypeptide of 465 amino acids, M(r) = 52,554. The centrally located catalytic domain contains all the conserved residues characteristic of eukaryotic protein kinases, with greatest similarity to the catalytic domains of 70-kDa ribosomal S6 protein kinase, protein kinase C, and protein kinase A. The C-terminal 75 residues also show homology to protein kinase C and S6 protein kinase. In contrast, the N-terminal 130 residues have no homology to any known protein, and thus may represent a new class of protein kinase regulatory domain. Other motifs found in the Atpk1 protein include two putative autophosphorylation sites, a pseudosubstrate site, two acidic domains, a lysine-rich domain, and two putative PEST sequences, which may contribute to the regulation of protein kinase activity. RNA-blot hybridization showed that atpk1 encoded a 1.8-kb mRNA. Analysis of atpk1 promoter/beta-glucuronidase reporter gene fusions in transgenic plants showed that atpk1 was expressed in all tissues and at all developmental stages, with the strongest expression observed in metabolically active tissues, suggesting that atpk1 is involved in the control of plant growth and development. The first intron of atpk1 functions as an enhancer in atpk1 expression.

Amino Acid Sequence↗
PubMed1994

atpk1, a novel ribosomal protein kinase gene from Arabidopsis. II. Functional and biochemical analysis of the encoded protein.

The Arabidopsis Atpk1 protein expressed in insect cells and plant cells exhibited multiple sizes consisting mainly of two doublets: p70 (68 and 70 kDa) and p85 (82 and 85 kDa). Extraction of p85 from cells required the presence of SDS, suggesting that p85 is associated with less soluble subcellular components. p70 was extracted by nonionic detergent without SDS, indicating that this form is cytoplasmic. p70 expressed in either Arabidopsis or insect cells underwent serine-specific autophosphorylation, indicating that Atpk1 is a protein-serine kinase. A point mutation (lysine 163 to arginine) in the ATP-binding site of the catalytic domain substantially diminished activity when expressed in insect cells. A 14-kDa protein (p14) was co-immunoprecipitated with p70 from insect cells expressing wild-type Atpk1 and was phosphorylated in immune complex kinase assays with Atpk1, suggesting it is a homolog of a natural substrate of Atpk1. Two plant ribosomal proteins (14 and 16 kDa) can be phosphorylated by the Atpk1 protein kinase, and we propose that Atpk1 is a novel ribosomal protein kinase. A 60-kDa form of Atpk1 derived from the insect cell-expressed p70 was more highly phosphorylated than p70 in in vitro kinase assays, suggesting a negative regulatory domain can be removed by proteolysis.

Animals↗
PubMed1994

Reappraisal of duplex criteria to assess significant carotid stenosis with special reference to reports from the North American Symptomatic Carotid Endarterectomy Trial and the European Carotid Surgery Trial.

PURPOSE: Duplex examination is today the principal initial method of assessing extracranial carotid or vertebral artery disease. However, varying haemodynamic criteria have been described to categorize the degree of internal carotid artery stenosis, and similarly the degree of stenosis detected with angiography has been assessed with different methods as highlighted in studies performed by the North American Symptomatic Carotid Endarterectomy Trial and the European Carotid Surgery Trial. This study looks at the correlation between two commonly used methods of assessing carotid artery stenosis with duplex criteria and the two methods of angiographic interpretation used in these trials. Duplex parameters are also identified to determine the greater than 70% stenosis group identified as at risk in these studies. METHODS: A total of 120 carotid bifurcations were studied in patients who underwent both carotid duplex and angiography. Correlations of duplex with angiography were assessed with duplex criteria described by Zwiebel and by Strandness and the angiographic methods used in studies performed by the North American Symptomatic Carotid Endarterectomy Trial and the European Carotid Surgery Trial. Receiver operator curves were constructed from the duplex data for the detection of stenosis greater than 70% based on the angiographic assessment used in the study performed by the North American Symptomatic Carotid Endarterectomy Trial. RESULTS: The duplex criteria described by Zwiebel and Strandness differed in their accuracy depending on which of the two methods was used to report the angiograms. Zwiebel's criteria agreed more with the angiographic method used in the study performed by the European Carotid Surgery Trial (sensitivity 98%, specificity 81%, accuracy 88%), whereas Strandness' criteria agreed more with the angiographic method used in the study performed by the North American Symptomatic Carotid Endarterectomy Trial (sensitivity 96%, specificity 85%, accuracy 89%). For the detection of a stenosis greater than 70%, a peak systolic velocity greater than 270 cm/sec and end diastolic velocity greater than 110 cm/sec provided a sensitivity of 96%, specificity of 91%, and accuracy of 93%. CONCLUSIONS: The accuracy of duplex studies compared with angiography in the assessment of extracranial vascular disease depends on the method of angiographic determination of carotid stenosis. Vascular laboratories should validate the duplex criteria they use against a standard method of angiographic assessment of carotid artery stenosis, with special reference to the recently reported studies noting the significance of a stenosis greater than 70% in patients with symptoms.

Blood Flow Velocity↗
PubMed1994

Direct in vivo visualization of right cardiac anatomy by fibreoptic endoscopy: observation of radiofrequency-induced acute lesions around the ostium of the coronary sinus.

Current mapping techniques used during electrophysiological study involve catheter placement under fluoroscopic guidance and are associated with prolonged radiation exposure. We considered that direct visualization of right heart anatomy by means of fibreoptic endoscopy could be useful in accurately localizing and guiding the ablation of arrhythmogenic substrates. Our goal was to evaluate the ability of this device to safely and accurately visualize the ostium of the coronary sinus and its vicinity as well as radiofrequency-induced acute lesions. Anaesthetized dogs (n = 4) were studied. Multipolar electrode catheters and a 3.6 mm diameter fibreoptic endoscope with a latex balloon covering the distal tip were inserted into the right atrium. The blood pressure, and surface and intracardiac electrocardiograms were recorded simultaneously. Radiofrequency energy was delivered just inside the coronary sinus ostium or in its vicinity. The acute lesions were carefully observed by endoscopy. Postmortem examination was then performed. With a balloon volume of 7-10 ml, the visual field was 15-20 mm in diameter. The blood pressure was generally stable. The ostium of the coronary sinus and its vicinity were clearly and accurately identified, and catheter placement in the coronary sinus and its vicinity could be achieved under direct vision in all four dogs. Additionally, the process by which acute lesions were created by radiofrequency was also directly visualized in great detail. There was a good concordance between endoscopically observed images and postmortem findings, and there was a strong correlation between delivered energy and the volume of the radiofrequency-induced lesions (r = 0.895).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Radiofrequency ablation for the fast pathway of atrioventricular node reentry. Evidence for partial damage to the fast and lower common pathways.

Radiofrequency ablation was attempted in a 30-year-old woman with atrioventricular (AV) node reentry of the common variety. Energy was delivered to ablate a fast pathway. After energy delivery, the atrio-His interval prolonged following transient AV node block. Retrograde conduction was no longer present. However, dual AV nodal physiology and AV node reentry with similar retrograde atrial activation sequence could be shown post-ablation. These observations suggest that both fast and lower common pathways were partially damaged by ablation.

Adult↗
PubMed1992

Spatial pattern of cdc2 expression in relation to meristem activity and cell proliferation during plant development.

The p34 protein kinase encoded by the cdc2 gene is a key component of the eukaryotic cell cycle required for the G1- to S-phase transition and entry into mitosis. To study the regulation of plant meristem activity and cell proliferation, we have examined the tissue-specific accumulation of cdc2 transcripts in Arabidopsis thaliana and the related crucifer radish (Raphanus sativus) by in situ hybridization using A. thaliana cdc2 cDNA sequences as a probe. cdc2 transcripts accumulated in leaf primordia and within the vegetative shoot apical meristem. During flower development, high levels of expression were observed in meristems, in the basal regions of developing organs, in the developing vasculature, and associated with rib meristems elaborated late in the development of some floral organs. In root tips, cdc2 transcripts accumulated in the meristematic region and adjacent daughter cells but were not detected in the quiescent center. There was strong hybridization throughout the pericycle, and a further localized accumulation of cdc2 transcripts was observed in the initial stages of the activation of a new meristem at sites of lateral root development. We conclude that cdc2 expression is a critical factor in the regulation of meristem activity and establishment of proliferative competence.

Base Sequence↗
PubMed1991

Characterization of a nuclear protein that binds to three elements within the silencer region of a bean chalcone synthase gene promoter.

The chalcone synthase (EC 2.3.1.74) gene promoter from the bean Phaseolus vulgaris L. contains a silencer element between positions -140 and -326 fro the transcription start site that is functional in electroporated soybean protoplasts. This element contains three binding sites for a bean nuclear factor (SBF-1) with DNA sequence recognition properties that are very similar to those of nuclear factor GT-1. By using a synthetic tetramer of one of the binding sites as probe, we have purified sequence-specific SBF-1 activity approximately 1750-fold from suspension-cell nuclei, by using a combination of ammonium sulfate precipitation, gel filtration, heparin-agarose chromatography, and sequence-specific DNA affinity chromatography. The factor exhibited an apparent molecular weight of 160,000-200,000 on the basis of gel filtration. A subunit molecular weight of approximately 95,000 was determined from SDS/polyacrylamide gel electrophoretic analysis of purified fractions, followed by Southwestern blot analysis (a protein blot probed with oligonucleotide probes), and from UV-cross-linking experiments. The factor lost DNA-binding activity on treatment with alkaline phosphatase. We discuss the properties of SBF-1 in relation to the functionality of GT-1 binding sequences in plant genes.

Acyltransferases↗
PubMed1991

Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to - 173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyl-transferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗
PubMed1990

Hydroxyproline-Rich Glycoprotein Transcripts Exhibit Different Spatial Patterns of Accumulation in Compatible and Incompatible Interactions between Phaseolus vulgaris and Colletotrichum lindemuthianum.

The distribution of transcripts encoding hydroxyproline-rich glycoproteins in hypocotyls of Phaseolus vulgaris L. infected with Colletotrichum lindemuthianum was examined by in situ hybridization to tissue sections. The expression of hypersensitive resistance in an incompatible interaction was accompanied by a massive early accumulation of transcripts in the epidermal, cortical, and perivascular parenchymal tissues immediately adjacent to the inoculation site. In a compatible interaction, there was no accumulation of transcripts in the epidermal and cortical tissues even though fungal hyphae ramified throughout these tissues. However, transcripts accumulated at a later stage in the perivascular tissue directly below the site of infection and in tissue several millimeters from the inoculation site. Thus, there is a spatial and tissue-specific counterpart to the differential timing of transcript accumulation in incompatible versus compatible interactions (AM Showalter, JN Bell, CL Cramer, JA Bailey, CJ Lamb [1985] Proc Natl Acad Sci USA 82: 6551-6555). These differences in the spatial distribution and tissue specificity of transcript accumulation imply the differential induction of signaling systems involved in race:cultivar-specific interactions.

Journal Article↗
PubMed1989

Molecular cloning of plant transcripts encoding protein kinase homologs.

Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences.

Amino Acid Sequence↗
PubMed1988

Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts.

To investigate the mechanisms underlying activation of plant defenses against microbial attack we have studied elicitor regulation of a chimeric gene comprising the 5' flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bacterial chloramphenicol acetyltransferase gene. Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled that of endogenous chalcone synthase genes in suspension cultured cells. Functional analysis of 5' deletions suggests that promoter activity is determined by an elicitor-regulated activator located between the "TATA box" and nucleotide position -173 and an upstream silencer between -173 and -326. These cis-acting elements function in the transduction of the elicitation signal to initiate elaboration of an inducible defense response.

Journal Article↗
PubMed1988

Glutathione causes a massive and selective induction of plant defense genes.

The reduced form of glutathione (GSH), when supplied to suspension cultured cells of bean (Phaseolus vulgaris L.) at concentrations in the range 0.01 to 1.0 millimolar, stimulates transcription of defense genes including those that encode cell wall hydroxyproline-rich glycoproteins and the phenylpropanoid biosynthetic enzymes phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) involved in lignin (PAL) and phytoalexin (PAL, CHS) production. Transcriptional activation of these genes leads to marked accumulation of the corresponding transcripts, contributing to a massive change in the overall pattern of protein synthesis which closely resembles that previously observed in response to fungal elicitor. GSH causes a marked increase in extractable PAL activity, whereas the oxidized form of glutathione, constituent amino acids, or other reducing agents are inactive. Possible roles of GSH in signaling biological stress are discussed.

Journal Article↗
PubMed1987

Transcriptional activation of plant defense genes by fungal elicitor, wounding, and infection.

Activation of plant defense genes was investigated by analysis of transcripts completed in vitro by isolated nuclei. Elicitor treatment of suspension-cultured bean (Phaseolus vulgaris L.) cells caused marked transient stimulation of transcription of genes encoding apoproteins of cell wall hydroxyproline-rich glycoproteins (HRGP) and the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), concomitant with the onset of rapid accumulation of the respective mRNAs and hence expression of the phytoalexin (PAL, CHS), lignin (PAL), and HRGP defense responses. While there was a lag of 2 h prior to stimulation of HRGP gene transcription, induction of the transcription of PAL and CHS genes occurred within 5 min of elicitor treatment. Induction of transcription of PAL, CHS, and HRGP genes was also observed in wounded hypocotyls and in infected hypocotyls during race-cultivar-specific interactions with the fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. Transcriptional activation occurred not only in directly infected tissue but also in distant, hitherto uninfected tissue, indicating intercellular transmission of an endogenous signal for defense gene activation. It is concluded that transcriptional activation of defense genes characteristically underlies induction of the corresponding defense responses and expression of disease resistance.

Cell Nucleus↗
PubMed1983

Elicitor induction of mRNA activity. Rapid effects of elicitor on phenylalanine ammonia-lyase and chalcone synthase mRNA activities in bean cells.

Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total pool of cellular mRNA.

Acyltransferases↗