Biomedical subjects

D A Christopher

Publications and source records attributed to D A Christopher.

At least 19 recordsLinked to original sources

PubMed2002

Characterization of three PDI-like genes in Physcomitrella patens and construction of knock-out mutants.

Plant genomes typically contain several sequences homologous to protein disulfide isomerase (PDI). PDI was first identified as an abundant enzyme in the endoplasmic reticulum, where it catalyzes the formation, reduction, and isomerization of disulfide bonds during protein folding. PDI-like proteins have also been implicated in a variety of other functions, such as the regulation of cell adhesion, and may act as elicitors of the autoimmune response in mammals. A PDI-like protein (RB60) was recently shown to be imported into chloroplasts in the unicellular green alga Chlamydomonas reinhardtii and a higher plant, Pisum sativum, where it associates with thylakoid membranes. This suggests that the different PDI-like proteins in plant and animals may have diverse biological roles. To begin to elucidate the roles of PDI-like proteins, we have cloned, characterized, and generated knock-out mutants for three PDI-like genes that have high, medium, and low levels of expression, respectively, in the moss Physcomitrella patens. Phylogenetic analysis indicates that the three PDI-like proteins cluster with RB60 and four proteins from Arabidopsis thaliana. They are typified by an N-terminal domain rich in negatively charged residues. The knock-out mutants, which are the first knock-outs available for PDI-like proteins in a multicellular organism, were found to be viable, indicating that the function of each single gene is dispensable, and suggesting that they may be functionally complementary.

Amino Acid Sequence↗

PubMed2001

RNA binding-proteins interact specifically with the Arabidopsis chloroplast psbA mRNA 5' untranslated region in a redox-dependent manner.

The 5' untranslated region (5'UTR) of the psbA mRNA (psbA encodes the PSII reaction center protein, D1) is a key site for RNA-protein interactions in the post-transcriptional regulation of gene expression. In this study, we mapped the major psbA mRNA 5'-terminus at -77 nt, and two minor termini clusters centered at -48 and -64 nt, upstream from the psbA translational start codon of Arabidopsis thaliana. RNA mobility shift, RNase protection and UV-crosslinking assays were used to characterize the interaction of chloroplast proteins with the RNA 5'UTR. RNA-protein interactions depended upon a thermolabile secondary structure and specific sequences in a 35 nt region of the 5'UTR, which were 80% conserved with the psbA 5'UTRs from five other plants. Major and minor proteins of 43- and 30-kDa, respectively, were detected by UV-crosslinking to RNA. Oxidizing conditions abolished the association of the proteins with the 5'UTR, while RNA-binding activity was recovered upon incubation with a reductant. Based on these findings, we hypothesize that post-transcriptional regulation of psbA gene expression in chloroplasts of vascular plants involves redox-dependent interactions between specific sequences in the 5'UTR and 43- and 30-kDa RNA-binding proteins.

5' Untranslated Regions↗

PubMed2001

Phytochrome A mediates blue light and UV-A-dependent chloroplast gene transcription in green leaves.

We characterized the photobiology of light-activated chloroplast transcription and transcript abundance in mature primary leaves by using the following two systems: transplastomic promoter-reporter gene fusions in tobacco (Nicotiana tabacum), and phytochrome (phyA, phyB, and hy2) and cryptochrome (cry1) mutants of Arabidopsis. In both dicots, blue light and UV-A radiation were the major signals that activated total chloroplast and psbA, rbcL, and 16S rrn transcription. In contrast, transcription activities in plants exposed to red and far-red light were 30% to 85% less than in blue light/UV-A, depending on the gene and plant species. Total chloroplast, psbA, and 16S rrn transcription were 60% to 80% less in the Arabidopsis phyA mutant exposed to blue light/UV-A relative to wild type, thus definitively linking phyA signaling to these photoresponses. To our knowledge, the major role of phyA in mediating the blue light/UV-A photoresponses is a new function for phyA in chloroplast biogenesis at this stage of leaf development. Although rbcL expression in plants exposed to UV-A was 50% less in the phyA mutant relative to wild type, blue light-induced rbcL expression was not significantly affected in the phyA, phyB, and cry1 mutants. However, rbcL expression in blue light was 60% less in the phytochrome chromophore mutant, hy2, relative to wild type, indicating that another phytochrome species (phyC, D, or E) was involved in blue light-induced rbcL transcription. Therefore, at least two different phytochromes, as well as phytochrome-independent photosensory pathways, mediated blue light/UV-A-induced transcription of chloroplast genes in mature leaves.

Arabidopsis↗

PubMed2001

Cryptochrome 1, cryptochrome 2, and phytochrome a co-activate the chloroplast psbD blue light-responsive promoter.

The reaction center core of photosystem II is composed of two chlorophyll binding proteins, D1 and D2, that are encoded by the chloroplast genes psbA and psbD. These chlorophyll binding proteins are damaged during photochemistry, especially under high irradiance. Photosystem II function is maintained under these conditions through turnover and resynthesis of D1 and D2. Blue light-activated transcription of psbD from a special light-responsive promoter is part of the repair system. In this study, light-activated chloroplast and psbD transcription were studied after dark adaptation of 21-day-old light-grown Arabidopsis plants. Illumination of dark-adapted plants with red light increased chloroplast transcription activity and transcription from the psbD light-responsive promoter. Blue light further increased chloroplast transcription activity and stimulated differential transcription from the psbD light-responsive promoter. Photoreceptor mutants showed that blue light-specific activation of chloroplast transcription and the psbD light-responsive promoter involve cryptochrome 1 (cry1) or cryptochrome 2 (cry2) and phytochrome A (phyA). Blue light-induced activation of the psbD light-responsive promoter was normal in det2-1 and hy5-1 but attenuated in det3-1. Therefore, cry1/cry2/phyA-mediated blue light activation of the psbD light-responsive promoter in 21-day-old Arabidopsis plants does not involve hy5, a transcription factor that mediates other phyA and blue light-induced responses.

Arabidopsis↗

PubMed2000

Advances in ultrasound biomicroscopy.

The visualisation of living tissues at microscopic resolution is attracting attention in several fields. In medicine, the goals are to image healthy and diseased tissue with the aim of providing information previously only available from biopsy samples. In basic biology, the goal may be to image biological models of human disease or to conduct longitudinal studies of small-animal development. High-frequency ultrasonic imaging (ultrasound biomicroscopy) offers unique advantages for these applications. In this paper, the development of ultrasound biomicroscopy is reviewed. Aspects of transducer development, systems design and tissue properties are presented to provide a foundation for medical and biological applications. The majority of applications appear to be developing in the 40-60-MHz frequency range, where resolution on the order of 50 microm can be achieved. Doppler processing in this frequency range is beginning to emerge and some examples of current achievements will be highlighted. The current state of the art is reviewed for medical applications in ophthalmology, intravascular ultrasound, dermatology, and cartilage imaging. Ultrasound biomicroscopic studies of mouse embryonic development and tumour biology are presented. Speculation on the continuing evolution of ultrasound biomicroscopy will be discussed.

Animals↗

PubMed2000

High-frequency color flow imaging of the microcirculation.

The extension of ultrasound (US) color flow imaging (CFI) techniques to high frequencies (> 20 MHz) has the potential to provide valuable noninvasive tools for scientific and clinical investigations of blood flow in the microcirculation. We describe the development of a slow-scan CFI system operating in the 20-100-MHz range that has been optimized to image the microcirculation. The apparatus has incorporated elements of a previously reported pulsed-wave Doppler system and is capable of operating in either CFI or pulsed-wave mode. The performance of the CFI system was evaluated at a center frequency of 50 MHz using two PVDF transducers with -6-dB beam widths of 43 and 60 microm. The -6 dB-axial resolutions were estimated to be 66 and 72 microm, respectively. In vivo validation experiments conducted using the murine ear model demonstrated the detection of flow in vessels down to 15-20 microm in diameter with flow velocities on the order of mm per s. Further experiments examining experimental murine tumors confirmed the successful detection of flow in the tumor microcirculation.

Animals↗

PubMed2000

Ultrasound for the visualization and quantification of tumor microcirculation.

Advances in ultrasound based methods for the non-invasive assessment of the tumor microcirculation are described. Two new ultrasound approaches are highlighted. The first method relies on commercially available ultrasound contrast agents in combination with specialized nonlinear imaging sequences. Nonlinear scattering by microbubble contrast agents provides a unique intravascular signature that can be distinguished from the echoes caused by surrounding tissues. Through destruction-reperfusion experiments it is possible to use microbubble contrast agents as a tracer revealing the kinetics of tumor blood flow. The second ultrasound method for examining the microcirculation involves the use of much higher frequencies. At frequencies on the order of 50 MHz, Doppler processing allows the direct assessment of flow dynamics in realtime in arterioles as small as 15 microm. Three dimensional Doppler maps of flow patterning are presented. The strengths and weaknesses of these new methods are discussed and the potential for their use in preclinical animal drug studies, clinical drug trials, and prognostic studies is described.

Animals↗

PubMed1999

Expression of a higher-plant chloroplast psbD promoter in a cyanobacterium (Synechococcus sp. strain PCC7942) reveals a conserved cis-element, designated PGT, that differentially interacts with sequence-specific binding factors during leaf development.

The chloroplast psbD gene, which encodes the D2 subunit of photosystem II, is regulated by a blue light-responsive promoter (BLRP). We tested the ability of different regions of the barley (Hordeum vulagare) BLRP to drive transcription of the lacZ reporter gene in genomic transformants of Synechococcus sp. strain PCC7942. The barley BLRP was transcribed in Synechococcus from the same initiation sites that are used in plant chloroplasts in vivo. A region of the BLRP, residing between -83 and -112 bp upstream from the transcription initiation sites, functioned as a negative element in Synechococcus. Nucleotide sequences within this region are conserved among the psbD genes of several monocots and dicots, and with the nuclear negative regulatory element GT. Thus this new cis-element was designated Plastid GT, PGT. Proteins from chloroplasts of barley and Arabidopsis thaliana interacted with PGT in a sequence-specific and developmental-dependent manner. The DNA-protein complexes from Arabidopsis chloroplasts are composed of 60- and 38-kDa polypeptides. We postulate that GT and PGT have evolved in the nucleus and chloroplast, respectively, from a common ancestral regulatory element.

Arabidopsis↗

PubMed1999

High-fluence blue light stimulates transcription from a higher plant chloroplast psbA promoter expressed in a cyanobacterium, Synechococcus (sp. strain PCC7942).

High-fluence white and blue light, but not red light, enhanced transcription of the barley chloroplast psbA promoter when heterologously expressed as a lacZ transcriptional fusion in the cyanobacterium, Synechococcus sp. strain PCC7942. Analysis of Arabidopsis thaliana phytochrome mutants, phyAphyB and hy2, indicated that a distinct blue phototransduction pathway stimulates psbA expression. The evolutionary implications of these findings are discussed.

Arabidopsis↗

PubMed1999

ADP-Dependent phosphorylation regulates association of a DNA-binding complex with the barley chloroplast psbD blue-light-responsive promoter.

The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a -10 promoter element and an activating complex, AGF, that binds immediately upstream of -35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between -71 and -100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1-5 mM) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mM), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.

Adenosine Diphosphate↗

PubMed1998

High-frequency Doppler ultrasound examination of blood flow in the anterior segment of the eye.

PURPOSE: To show that extending Doppler imaging into the high-frequency domain could allow detection and characterization of blood flow in small arterioles and capillaries. METHODS: A 40-MHz continuous wave Doppler system and a 60-MHz pulsed-wave Doppler system were constructed, tested, and used to examine the ciliary body region in two normal volunteers. RESULTS: Ciliary body circulation in the region of the great circle of the iris, which is undetectable by conventional 7.5-MHz duplex Doppler, was consistently and reproducibly detectable by high-frequency (40-MHz and 60-MHz) Doppler systems. CONCLUSION: High-frequency Doppler imaging may provide a unique new tool for the characterization and assessment of anterior segment ocular blood flow.

Anterior Eye Segment↗

PubMed1998

40-MHZ echocardiography scanner for cardiovascular assessment of mouse embryos.

Congenital heart disease results from genetic defects that are manifested at early stages of embryogenesis. The mouse is the preferred animal model for studies of mammalian embryonic development and for an increasing number of human disease models. A number of genes identified in the mouse are critical for normal cardiovascular development, but an understanding of the underlying mechanisms regulating heart development is still incomplete, in part because of the lack of methods to measure hemodynamics in live mouse embryos. We describe the development of a 40-MHz ultrasound scanner, which allows image-guided continuous-wave and pulsed Doppler blood flow measurements in mouse embryos, in utero, at the critical early developmental stages. Doppler waveforms acquired from mouse embryonic umbilical vessels, descending aorta, and cardiac ventricles are presented to demonstrate the utility of the method. By combining image-guided ultrasound Doppler with the many available mouse mutants, this approach should lead to new insights into embryonic cardiovascular structure-function relationships.

Animals↗

PubMed1998

DET1 represses a chloroplast blue light-responsive promoter in a developmental and tissue-specific manner in Arabidopsis thaliana.

The chloroplast psbD-psbC loci, which encode the D2 and CP43 subunits of the photosystem II reaction center, respectively, are regulated by a blue light-responsive promoter (BLRP). It has recently been shown in barley seedlings that activation of psbD-psbC transcription by blue light involves inhibition of a protein kinase that represses the BLRP in the dark. To elucidate further the photosensory pathways regulating the psbD BLRP, the effects of three nuclear mutations on the expression of the BLRP in chloroplasts of Arabidopsis thaliana were examined. The mutants used included the det1-1 and det1-6 alleles for the nuclear protein DET1, involved in repressing photomorphogenesis, and the cry1 gene for the blue light photoreceptor, cryptochrome (CRY1), involved in hypocotyl elongation. The BLRP was not significantly expressed in cotyledons of light-grown wild-type seedlings, unlike the light-responsive expression of the chloroplast, psbA and rbcL, and nuclear, Lhcb and Chs, genes. Analysis of the mutants revealed that DET1 represses transcription from the BLRP in a developmental and tissue-specific manner, which is unique from the effects that DET1 has on other light-regulated promoters. In addition, the cry1 mutation did not reduce the expression of the BLRP in response to blue light. This suggests that the BLRP is regulated by a different photosensory system relative to CRY1. A model is proposed involving blue light, DET1 and phytochrome in regulating transcription from the psbD BLRP.

Arabidopsis↗

PubMed1997

A high-frequency pulsed-wave Doppler ultrasound system for the detection and imaging of blood flow in the microcirculation.

Previous work with a 40-MHz continuous-wave Doppler ultrasound system has demonstrated the potential of high-frequency Doppler ultrasound (HFD), operating in the frequency range 20-200 MHz, to detect blood flow in the microcirculation. This paper describes a directional, pulsed-wave high-frequency Doppler ultrasound (PW HFD) system that was designed and constructed further to investigate this potential. The PW HFD system electronics have a dynamic range of > 80 dB, a noise floor of 250 nV, a directional isolation of 45 dB and operate over the frequency range 1-200 MHz. The system is tested using a focused PVDF transducer that is tuned for maximum sensitivity at 50 MHz, has a -6 dB lateral beamwidth of 70 microns and -6 dB depth-of-focus of 0.90 mm. This permits the practical use of sample volumes with dimensions 70 microns laterally by 90-900 microns axially. The PW HFD system can operate in duplex mode by either sharing the Doppler transducer or using a second PVDF transducer for imaging. Tests with string and capillary flow phantoms demonstrate that the PW HFD system is capable of detecting velocities on the order of the blood velocities found in the capillaries (0.5 mm/s) and arterioles (5 mm/s) with suitable velocity (30-300 microns/s) and temporal (15-100 ms) resolutions. In vivo measurements demonstrate that PW HFD can detect and measure blood velocities of less than 5 mm/s in arterioles and venules with diameters as small as 20 microns and 35 microns, respectively, using a sample volume of only 70 microns laterally by 150 microns axially. Preliminary experiments with high-frequency colour Doppler (HFCD) and high-frequency power Doppler (HFPD) imaging are also presented.

Animals↗

PubMed1997

Involvement of protein kinase and extraplastidic serine/threonine protein phosphatases in signaling pathways regulating plastid transcription and the psbD blue light-responsive promoter in barley.

We investigated the signaling pathways that control changes in plastid transcription in response to development and light. Plastid gene expression was analyzed in dark-grown barley (Hordeum vulgare L.) seedlings treated in vivo with an inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), or an inhibitor of protein kinases (K252a), followed by exposure of the seedlings to either red, blue, or white light. OA prevented blue light from activating the plastid pshD blue-light-responsive promoter (BLRP) and prevented red and blue light from activating the expression of the plastid-encoded rbcl and psbA and the nuclear-encoded RbcS and Lhcb genes. OA reduced total plastid transcription activity in dark- and light-grown seedlings by 77 to 80%, indicating that OA prevented light-responsive transcription by reducing total plastid transcription. In contrast, K252a activated the accumulation of mRNAs arising from the BLRP. Blue light in combination with K252a increased psbD mRNA levels in an additive manner. The results indicate that protein phosphatases 1 and/or 2A, which reside external to the organelle, are required for proper function of plastid transcription and chloroplast development, whereas a protein kinase represses the BLRP in plants grown in the dark.

Carbazoles↗

PubMed1997

Structure and blue-light-responsive transcription of a chloroplast psbD promoter from Arabidopsis thaliana.

We characterized the effects of light on psbD transcription and mRNA levels during chloroplast development in Arabidopsis thaliana. After 6 to 12 hours of illumination of dark-grown seedlings, two psbD mRNAs were detected and their 5' ends were mapped to positions -550 and -190 bp upstream from the psbD translational start codon. Their kinetics of accumulation resembled the accumulation of chloroplast psbA and rbcL mRNAs but differed from the accumulation of the nuclear-encoded Lhcb and Chs mRNAs. A third psbD mRNA with its 5' ends at position -950 accumulated after illumination of > 180 h. The 5' ends of this transcript were mapped to a nucleotide sequence that is highly conserved with functional sequences in the barley (Hordeum vulgare) blue-light-responsive promoter (BLRP). Transcription from the Arabidopsis psbD promoter was 3-fold higher in blue relative to red light, whereas red and blue light affected total chloroplast, rbcL, and 16S rDNA transcription similarly. This study shows that transcription of Arabidopsis psbD is mediated by a BLRP and suggests that psbD genes in other land plants are regulated by a common blue-light-signaling pathway. Isolating the BLRP from Arabidopsis will allow molecular genetic studies aimed at identifying the pertinent photoreceptor and components of this phototransduction pathway.

Adaptation, Physiological↗

PubMed1996

A high-frequency continuous-wave Doppler ultrasound system for the detection of blood flow in the microcirculation.

Basic ultrasound physics and several clinical and experimental observations suggest that high-frequency Doppler ultrasound (HFD) operating in the frequency range 20-100 MHz holds the promise of detecting blood flow in the microcirculation. This article describes a directional, continuous-wave (CW), 1- to 200-MHz Doppler ultrasound system. The system electronics have a dynamic range of 100 dB, a noise floor of 10 nV and a directional isolation of 50 dB. The development of a 40-MHz Doppler transducer composed of two, 81-micron-thick, lithium niobate crystals that have been air-backed and transmission-line tuned for maximum sensitivity is described. This device is used to test the CW Doppler system using string and capillary phantoms and in vivo tissue. We show that HFD can detect and measure velocities on the order of the blood velocities found in the capillaries (1 mm/s) and arterioles (5 mm/s) with suitable velocity (50-500 microns/s) and temporal (20-250 ms) resolutions. In vivo measurements demonstrate that HFD is sensitive to the detection of blood flow in small vessels.

Arterioles↗

PubMed1995

A wall-less vessel phantom for Doppler ultrasound studies.

Doppler ultrasound flow measurement techniques are often validated using phantoms that simulate the vasculature, surrounding tissue and blood. Many researchers use rubber tubing to mimic blood vessels because of the realistic acoustic impedance, robust physical properties and wide range of available sizes. However, rubber tubing has a very high acoustic attenuation, which may introduce artefacts into the Doppler measurements. We describe the construction of a wall-less vessel phantom that eliminates the highly attenuating wall and reduces impedance mismatches between the vessel lumen and tissue mimic. An agar-based tissue mimic and a blood mimic are described and their acoustic attenuation coefficients and velocities are characterised. The high attenuation of the latex rubber tubing resulted in pronounced shadowing in B-mode images; however, an image of a wall-less vessel phantom did not show any shadowing. We show that the effects of the highly attenuating latex rubber vessels on Doppler amplitude spectra depend on the vessel diameter and ultrasound beam width. In this study, only small differences were observed in spectra obtained from 0.6 cm inside diameter thin-wall latex, thick-wall latex and wall-less vessel phantoms. However, a computer model predicted that the spectrum obtained from a 0.3-cm inside diameter latex-wall vessel would be significantly different than the spectrum obtained from a wall-less vessel phantom, thus resulting in an overestimation of the average fluid velocity. These results suggest that care must be taken to ensure that the Doppler measurements are not distorted by the highly attenuating wall material. In addition, the results show that a wall-less vessel phantom is preferable when measuring flow in small vessels.

Acoustics↗