The mechanism of peripheral recanalization by laser-assisted thermal angioplasty: confirmation by intravascular sonography.
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Aged↗
Biomedical subjects
J Griffith
Publications and source records attributed to J Griffith.
Human genotoxicity in phosphine-exposed fumigant applicators.
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Chromosome Aberrations↗
Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy.
Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.
Adenosine Triphosphate↗
Human genotoxicity: pesticide applicators and phosphine.
Fumigant applicators who, 6 weeks to 3 months earlier, were exposed to phosphine, a common grain fumigant, or to phosphine and other pesticides had significantly increased stable chromosome rearrangements, primarily translocations in G-banded lymphocytes. Less stable aberrations including chromatid deletions and gaps were significantly increased only during the application season, but not at this later time point. During fumigant application, measured exposure to phosphine exceeds accepted national standards. Because phosphine is also used as a dopant in the microchip industry and is generated in waste treatment, the possibility of more widespread exposure and long-term health sequelae must be considered.
Chromosome Aberrations↗
Molecular cloning, characterization, and expression of a human 14-kDa lectin.
Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.
Amino Acid Sequence↗
Trigeminal nerve section induces Fos-like immunoreactivity (FLI) in brainstem and decreases FLI in sensory cortex.
Transecting the infraorbital nerve to the rat whiskers induced Fos-like immunoreactivity (FLI) in lamina I and II neuronal nuclei of the spinal trigeminal nucleus pars caudalis (Sp5c). The Fos-like immunostaining persisted for several weeks. The prolonged expression of FLI in Sp5c could be related to persistent activity in the sectioned nerve, or to trophic effects of injured ganglion neurons on brainstem cells. We postulate that Fos and related proteins may be involved in mediating alterations in gene expression associated with relatively long-term CNS adaptations to peripheral nerve injuries. Surprisingly, FLI decreased in contralateral sensory cortex, mainly in layers 2, 3 and 6, up to several days after the lesion. These decreases of cortical FLI may be due to decreased sensory neuronal activity, and/or to reducing the trophic influence of thalamic inputs on cortical neurons.
Animals↗
Antibiotic proteins of human polymorphonuclear leukocytes.
Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.
Amino Acid Sequence↗
Hyperacute rejection of the transplanted mouse heart.
Mouse hearts transplanted heterotopically to MHC-disparate recipients can be hyperacutely rejected (HAR) after a single or 3 sequential donor type skin grafts, or a single intradermal injection of lymphoid cells. In the combinations tested, not all hearts are HAR; most of them are rejected in accelerated fashion. Our results with transplanted rat hearts are similar, even in a genetic combination for which HAR of all hearts has been reported. However, in rats, HAR tends to occur more rapidly and to be associated with more-intense vascular changes. Transfer of serum from mice or rats sensitized by 3 sequential skin grafts likewise resulted in occasional hyperacute but never accelerated rejection. Transfer of lymph node cells from mice sensitized with a single skin graft always resulted in accelerated but never in hyperacute rejection; transfer of cells after 3 sequential skin grafts caused neither accelerated nor hyperacute rejection.
Acute Disease↗
Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis.
A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.
DNA Repair↗
Intravascular ultrasound cross-sectional arterial imaging before and after balloon angioplasty in vitro.
A prototype ultrasound imaging catheter was evaluated in vitro using 17 human atherosclerotic artery segments before and after balloon dilatation angioplasty. The catheter was 1.2 mm in diameter and incorporated a single 20-MHz ultrasound transducer to obtain cross-sectional images of the arterial lumen. In 15 of the 17 (88%) arteries, high quality images were obtained, which demonstrated clear demarcation between the lumen and the endothelium, the atheroma plaque, the muscular media, and the adventitia. Qualitative characteristics of plaque disruption, dissection, and residual flaps were readily visible. In addition, quantitative information about cross-sectional lumen area was obtained before and after balloon dilatation. The mean cross-sectional lumen area increased from 8.7 to 15.1 mm2 (p less than 0.01) following balloon dilatation. The lumen area measured from the ultrasound images following dilatation correlated closely with the area measured from histologic sections (r = 0.88). The results from this study indicate that a small-diameter ultrasound imaging catheter can be developed that will provide high-resolution qualitative and quantitative information during peripheral and coronary angioplasty.
Angioplasty, Balloon↗
Isolation of a complementary DNA clone encoding a precursor to human eosinophil major basic protein.
A 14-kD protein was purified from human PMNs and its NH2-terminal sequence was determined. Comparison of a portion of the NH2-terminal sequence of this protein to the recently reported NH2-terminal sequence of eosinophil major basic protein (MBP) showed them to be identical. To aid further characterization of the structural and functional properties of this molecule, we isolated from an HL-60 cDNA library a single class of cDNA clones whose sequence matched exactly the NH2-terminal amino acid sequence of the 14-kD polypeptide. Northern analysis of HL-60 cells suggests that MBP is constitutively expressed in HL-60 cells and is highly transcribed from a single copy gene. The sequence of the full-length cDNA clones predicts that MBP is synthesized as a 23-kD precursor form (pro-MBP) which is subsequently cleaved to release the mature 14-kD MBP. The putative pro-MBP has a predicted pI of 6.0, but both the charged and the hydrophobic residues are asymmetrically distributed, creating a bipolar molecule. The NH2-terminal half has a predicted pI of 3.7 and is hydrophilic, while the COOH-terminal half (corresponding to mature MBP) has a predicted pI of 11.1 and is hydrophobic.
Base Sequence↗
Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct.
We have investigated the structural change in a double-stranded DNA helix caused by covalent addition of a psoralen. A synthetic double-stranded DNA was constructed to contain either a psoralen furan-side monoadduct or an interstrand diadduct at a specific site. When the unmodified and psoralen modified DNAs were examined by electron microscopy in the presence of distamycin, which stiffens the DNA helix, the DNA containing the psoralen interstrand diadduct appeared bent (or kinked), whereas the furan-side monoadducted DNA appeared similar to the unmodified DNA. RecA protein from E. coli has been shown to preferentially bind UV (ultra violet) irradiated DNA presumably due to alterations in the normal DNA helical structure. Using a nitrocellulose filter binding assay, we have found that the psoralen interstrand diadduct enhances the binding of recA protein to the double-stranded DNA, whereas a furan-side monoadduct has little effect. Thus both the recA protein binding and the electron microscopic data suggest that a psoralen diadduct causes deformation of a DNA helix, most likely by kinking the helix, and that a monoadduct has little effect on the DNA helix structure.
Base Sequence↗
Direct visualization of RecA protein binding to and unwinding duplex DNA following the D-loop cycle.
The RecA protein of Escherichia coli will promote the plectonemic joining of a linear single-stranded DNA molecule with a homologous supertwisted double-stranded (ds) DNA molecule. As shown by others, this reaction is characterized by a single cycle of joint formation and dissociation, termed the D-loop cycle. The released DNA products appear by electron microscopy to be topologically identical to the reactant DNAs, yet a second cycle of joining is not observed. This implies that either the RecA protein-single-stranded DNA filament or the dsDNA must be altered during the pairing reaction such that further joint formation is inhibited. Shibata et al. (Shibata, T., DasGupta, C., Cunningham, R. P., Williams, J. K. G., Osber, L., and Radding, C. M. (1982) J. Biol. Chem. 256, 7565-7572) proposed that the dsDNA was inactivated due to the binding of RecA protein following the D-loop cycle, but were unable to describe the structure of the putative RecA-protein-dsDNA complex. Here we have extended those studies to show that if fresh dsDNA is added to a reaction mixture following completion of the D-loop cycle, joint formation is stimulated, but only with the freshly added dsDNA. Following completion of the D-loop cycle, a labile RecA protein-dsDNA complex, in which the dsDNA is partially unwound can be preserved by glutaraldehyde fixation and visualized by electron microscopy. This result provides direct evidence that the block to a second cycle of joining is due to the presence of RecA protein remaining bound to the released dsDNA.
DNA↗
Purification and visualization of native spliceosomes.
Mammalian spliceosomes were purified in preparative amounts by gel filtration chromatography and shown to be functional by in vitro complementation experiments. The column fractions containing spliceosomes are enriched in the snRNAs U1, U2, U4, U5, and U6 and a subset of proteins present in the nuclear extract. Splicing intermediates, the entire set of snRNAs, and the enriched proteins can be immunoprecipitated with three different monoclonal antibodies that recognize snRNP determinants. At least one U1 snRNP is present in each spliceosome since the particles are quantitatively immunoprecipitated by an anti-U1 snRNP monoclonal antibody. Examination of the spliceosome fractions by EM revealed a relatively homogeneous population of 40-60 nm particles with a striking morphology. Evidence that these particles are spliceosomes is their sensitivity to micrococcal nuclease, their ATP-dependent assembly, and their immunoprecipitation with a trimethyl cap monoclonal antibody. In addition, pre-mRNA was visualized in the particles by EM.
Adenosine Triphosphate↗
Thymine dimers bend DNA.
A 32-base-pair DNA fragment containing a thymine photodimer was constructed and ligated head-to-tail to obtain multimers of this sequence in which thymine dimers were in phase with the helix screw axis (approximately equal to 3 turns apart). The ligation products were analyzed by one- and two-dimensional gel electrophoresis and quantitative electron microscopy. These analyses show that the thymine photodimer introduces a bend of approximately equal to 30 degrees in DNA, which causes anomalously slow migration of DNA fragments in polyacrylamide gels and facilitates the formation of small covalent circles. Repair of thymine dimers by DNA photolyase abolishes the anomalous migration.
DNA↗
Successful single lung transplantation.
We have presented a successful case of lung transplantation in a man with pulmonary fibrosis. We believe it is the first successful attempt in the United States, following demonstration of its feasibility in Toronto, Canada. Twenty years after the first lung transplantation, several factors have evolved that help to secure a successful outcome, including a strong, dynamic organ procurement system; static, hypothermic lung preservation; omental wrapping of the bronchial anastomosis; improved immunosuppressive agents; and improved postoperative care.
Humans↗
Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange.
The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products.
Base Composition↗
Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein.
The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.
DNA, Bacterial↗