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Biomedical subjects

J M Burke

Publications and source records attributed to J M Burke.

At least 19 recordsLinked to original sources
PubMed1992

Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes.

The catalytic function of the hairpin ribozyme has been investigated by modification-interference analysis of both ribozyme and substrate, using ribonucleoside phosphorothioates. Thiophosphate substitutions in two ribozyme domains were examined by using a novel and highly efficient two-piece ribozyme assembled from two independently synthesized oligoribonucleotides. The catalytic proficiency of the two-piece construct (KM = 48 nM, kcat = 2.3 min-1) is nearly identical to that of the one-piece ribozyme. The two-piece ribozyme is essentially unaffected by substitution with thiophosphates 5' to all guanosines, cytidines, and uridines. In contrast, incorporation of multiple adenosine phosphorothioates in the 5' domain of the ribozyme decreases ribozyme activity by a factor of 25. Modification-interference experiments using ribozymes partially substituted with adenosine phosphorothioate suggest that thiophosphates 5' to A7, A9 and A10 interfere with cleavage to a greater extent than substitutions at other sites within the molecule, but the effect is modest. Within the substrate, phosphorothioate substitution does not directly interfere with cleavage, rather, increasing thiophosphate content decreases the stability of the ribozyme-substrate complex. We describe the construction of a hairpin ribozyme containing dinucleotide extensions at its 5' and 3' ends. Full substitution of this molecule with G and C phosphorothioates results in a ribozyme with greatly enhanced stability against cellular ribonucleases without significant loss of catalytic efficiency.

Base Sequence↗
PubMed1992

Modulation of potassium transport in cultured retinal pigment epithelium and retinal glial cells by serum and epidermal growth factor.

The ionic environment of retinal photoreceptors is partially controlled by potassium transporters on retinal glial and retinal pigment epithelial cells (RPE). In this study, serum and epidermal growth factor (EGF) were examined as modulators of potassium transport in confluent cultures of human RPE and rabbit retinal glia. EGF is a known mitogen for confluent RPE cultures and was shown here to also stimulate [3H]thymidine incorporation in cultures of retinal glia. For potassium transport studies 86Rb was used as a tracer during a 17-min incubation. For both retinal cell types the mean total 86Rb uptake in 10% serum was approximately 60% above basal, serum-free controls. For EGF, tested in several experiments in a concentration range from 1 to 100 ng/ml, maximal total uptake was 33 and 24% above controls for RPE and glia, respectively. Inhibitor studies suggested that basal and serum-stimulated uptake for both cell types occurred by the ouabain-sensitive Na-K ATPase pump and by the furosemide- or bumetanide-sensitive Na-K-Cl cotransporter. EGF-stimulated uptake appeared to be due predominantly to the cotransporter. The data suggest that serum components and EGF, which may be available to retina-derived cells under pathologic conditions, may not only stimulate proliferation but may also act as short-term modulators of potassium ion movement and thus affect physiologic processes that are sensitive to ion homeostasis.

Animals↗
PubMed1992

In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions.

In vitro selection methods provide rapid and extremely powerful tools for elucidating interactions within and between macromolecules. Here, we describe the development of an in vitro selection procedure that permits the rapid isolation and evaluation of functional hairpin ribozymes from a complex pool of sequence variants containing an extremely low frequency of catalytically proficient molecules. We have used this method to analyze the sequence requirements of two regions of the ribozyme-substrate complex: a 7-nucleotide internal loop within the ribozyme that is essential for catalytic function and substrate sequences surrounding the cleavage-ligation site. Results indicate that only 3 of the 16,384 internal loop variants examined have high cleavage and ligation activity and that the ribozyme has a strong requirement for guanosine immediately 3' to the cleavage-ligation site.

Base Composition↗
PubMed1991

Novel guanosine requirement for catalysis by the hairpin ribozyme.

THERE is much interest in the development of 'designer ribozymes' to target destruction of RNAs in vitro and in vivo. Engineering of ribozymes with novel specificities requires detailed knowledge of the ribozyme-substrate interaction, and a rigorous evaluation of sequence specificity. The hairpin ribozyme catalyses an efficient and reversible site-specific cleavage reaction. We have used mutagenesis and in vitro selection strategies to show that RNA cleavage and ligation has an absolute requirement for guanosine immediately 3' to the cleavage-ligation site. This G is not required for efficient substrate binding, rather, its 2-amino group is an essential component of the active site required for catalysis.

Base Sequence↗
PubMed1991

Binding and cleavage of nucleic acids by the "hairpin" ribozyme.

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.

Base Sequence↗
PubMed1991

Methanol poisoning. A rodent model with structural and functional evidence for retinal involvement.

Methanol ingestion can lead to visual impairment, central nervous system dysfunction, or death. The extent of ocular involvement has been difficult to determine because the toxicity is restricted to humans and nonhuman primates due to species differences in methanol metabolism. A rodent model of methanol toxicity recently developed by us was used to evaluate retinal dysfunction in methanol poisoning. Formic acidemia and visual toxic reactions developed in methanol-intoxicated rats. Electroretinographic analysis indicated a significant early deficit in b-wave amplitude followed by a temporally delayed, lesser reduction in a-wave amplitude. Histologic evaluation of the eyes 60 hours after methanol administration revealed generalized retinal edema and vacuolation in the photoreceptors and retinal pigment epithelium. Ultrastructural examination showed swelling and disruption of the mitochondria in photoreceptor inner segments, optic nerve, and the retinal pigment epithelium. These studies document direct retinal involvement in this nonprimate model of methanol toxicity.

Animals↗
PubMed1991

The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium.

The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [3H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [3H]thymidine and [3H]ouabain. Cycling cells which had [3H]thymidine-labeled nuclei did not have notably higher labeling with [3H]ouabain. However, [3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.

Autoradiography↗
PubMed1991

Retinal pigment epithelial cells of the posterior pole have fewer Na/K adenosine triphosphatase pumps than peripheral cells.

The density of Na/K adenosine triphosphatase (ATPase) pumps in retinal pigment epithelial (RPE) cells in different retinal regions was quantified by measuring the binding of 3H-ouabain to RPE in cow and human eyecups. In bovine eyes, pump density was estimated in RPE samples isolated from three retinal regions outlined with a 7-mm trephine: one from the posterior pole in the area centralis and two from the superior, equatorial retina representing unpigmented (in the tapetum) and pigmented zones. In human eyes, RPE samples were isolated from a posterior region centered around the macula and one superior region. Ouabain binding to RPE of the posterior pole of both species was approximately 40-60% lower than binding to RPE of more peripheral regions in the same eyes. For bovine eyes, ouabain binding did not differ between pigmented and unpigmented cells of the superior retina, suggesting that reduced binding in the relatively amelanotic posterior cells was not related to levels of pigmentation. For human RPE, binding to posterior cells was lower in eyes from donors of all ages (range, 17-90 yr). The data suggest that Na/K ATPase pump site density is lower in posterior RPE cells of both bovine and human eyes, perhaps due to a regional difference in requirements for ionic regulation.

Adolescent↗
PubMed1990

A 3' splice site-binding sequence in the catalytic core of a group I intron.

Ribozymes use specific RNA-RNA interactions for substrate binding and active-site formation. Self-splicing group I introns have approximately 70 nucleotides constituting the core, a region containing sequences and structures indispensable for catalytic function. The catalytic core must interact with the substrates used for the two steps of the self-splicing reaction, that is, guanosine, the 5'-splice-site helix (P1) and the 3' splice site. Mutational evidence suggests that core sequences near segment J6/7 that joins the base-paired stems P6 and P7, and the bulged base of P7(5'), participate in binding guanosine substrate, but nothing is known about the interactions between the core, the 5'-splice-site helix and the 3' splice site. On the basis of comparative sequence data, it has been suggested that two specific bases in the catalytic core of group I introns might form a binding sequence for the 3' splice site. Here we present genetic evidence that such a binding site exists in the core of the Tetrahymena large subunit ribosomal RNA intron. We demonstrate that this pairing, termed P9.0, is functionally important in the exon ligation step of self-splicing, but is not itself responsible for 3'-splice-site selection.

Animals↗
PubMed1990

Clearance of experimental vitreous hemorrhage after panretinal cryotherapy is related to macrophage influx.

We investigated the effect of panretinal cryotherapy on blood clearance and on the inflammatory cellular response in rabbit eyes with experimental vitreous hemorrhage. Eyes treated with cryotherapy demonstrated less extensive vitreous opacity and more rapid clearance of blood than untreated eyes. The peak inflammatory cellular response measured by counts or radiolabeled cells in the optic nerve was 2.5 times greater in the cryotherapy-treated eyes. Panretinal cryotherapy may promote the clearance of vitreous hemorrhage by stimulating the influx of increased numbers of phagocytic inflammatory cells.

Animals↗
PubMed1990

Modulation of growth in retina-derived cells by extracellular matrices.

Intravitreal membranes from patients with proliferative vitreoretinopathy (PVR) consist partly of retinal glial (RG) and retinal pigment epithelial (RPE) cells surrounded by varying amounts of extracellular matrix (ECM). The contribution of the ECM to the growth of PVR membranes is unknown. This study was undertaken to determine if proliferation in cultured RPE and RG cells is affected by different substrates, including some ECM materials which have been identified in PVR membranes. Substrates tested included type I collagen, basement membrane Matrigel, and poly-D-lysine, as well as uncharacterized cell type-specific matrices deposited by cultured RPE and RG cells. Proliferation was quantified by 3H-thymidine incorporation and radioautography 24 hours after plating and by cell counts after 14 days in the presence of serum. Relative to uncoated culture plastic, growth of RPE cells was inhibited by Matrigel, enhanced by poly-D-lysine, and unaffected by type I collagen. In contrast, growth in RG cells was inhibited by type I collagen and unaffected by the other substrates. Analysis of the timing of DNA synthesis after plating suggested that the substrates which affected RPE growth did so by altering the fraction of cycling cells rather than the cell cycle time. For the cell-derived matrices, heterotypic matrix (matrix produced by the other retinal cell type) enhanced the growth of both RPE and RG. The results suggest that the ECM may modify the growth of cells contributing to PVR membranes. Of note is that the cell-derived matrices reciprocally stimulated growth of RG and RPE cells, cell types which may interact in PVR membranes.

Animals↗
PubMed1989

Selection of the 3'-splice site in group I introns.

A model for selection of 3'-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3'-splice site. This tertiary pairing would bring the 3'-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3'-exon sequences and the 'internal guide sequence' near the 5'-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3'-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.

Introns↗
PubMed1989

Compensatory mutations demonstrate that P8 and P6 are RNA secondary structure elements important for processing of a group I intron.

Compensatory mutations have been constructed which demonstrate that P8 and P6, two of nine proposed base-pairing interactions characteristic of group I introns, exist within the folded structure of the Tetrahymena thermophila rRNA intervening sequence, and that these secondary structure elements are important for splicing in E. coli and self-splicing in vitro. Two-base mutations in the 5' and 3' segments of P8 are predicted to disrupt P8 and a strong splicing-defective phenotype is observed in each case. A compensatory four-base mutation in P8 is predicted to restore pairing, and results in the restoration of splicing activity to nearly wild type levels. Thus, we conclude that P8 exists and is essential for splicing. In contrast to the strong phenotypes generally exhibited by mutations which disrupt RNA secondary structure, a two-base mutation in L8, the loop between P8[5'] and P8[3'], results in only a slight decrease in splicing activity. We also tested P6, a pairing which is proposed to consist of only two base-pairs in this intron. A two-base mutation in P6[3'] reduces splicing activity to a greater extent than does a two-base mutation in P6[5']. Comparison of the activities of these mutants and a compensatory P6 four-base mutant support the existence of P6, and suggest that the P6 pairing may be particularly important in the exon ligation step of splicing.

Animals↗
PubMed1989

Photoablation of ocular melanoma with a high-powered argon endolaser.

We studied the use of a 15-W argon blue-green laser in the treatment of choroidal melanoma in a rabbit model. Greene melanoma cells were used to produce 2- to 4-mm thick tumors posteriorly in the suprachoroidal space in pigmented rabbits. Endophotocoagulation delivered through a 600-micron fiberoptic probe was performed to ablate the tumor tissue and a surrounding margin of normal tissue. A vitreous cutter was used simultaneously to remove liberated necrotic debris. The effect of the laser on tumor and normal ocular tissue was evaluated by light microscopy and the extent of the proliferative response by tritiated thymidine radioautography. Application of 100 to 400 pulses of laser energy using treatment parameters of 12 to 14 W of power and 0.1-s pulses resulted in complete ablation of melanoma tissue, overlying retina, and choroid. There was no substantial intraoperative or postoperative hemorrhage. Material liberated during the laser treatment was found to be nonviable. The effect of the laser on tissue appeared localized to within approximately 1.25 mm of the margin of the central lesion. The high-energy argon laser seems to offer a means of effectively ablating melanoma tissue via an internal resection approach.

Animals↗
PubMed1989

Growth in retinal glial cells in vitro is affected differentially by two types of cell contact-mediated interactions.

Contact among rabbit retinal glial cells in subconfluent culture was previously shown to stimulate DNA synthesis [J. M. Burke (1983) Exp. Cell Res. 146, 204-206]. In this study nonliving surface membranes and metabolic coupling were investigated as mediators of the contact-dependent phenomenon. To evaluate surface membranes, preparations of fixed glial cells and fixed fibroblasts of several types were added in varying numbers to sparse cultures of glia or fibroblasts. In agreement with published data, fibroblast proliferation was inhibited by the fixed cells in a dose-dependent manner. Growth in glial cells was similarly inhibited. Fixed cells of both types were approximately equally effective in suppressing proliferation in cells of both types. No number of fixed cells was identified which, when added to glial cultures, stimulated glial proliferation. In contrast, metabolic coupling among glial cells was associated with increased DNA synthesis. Coupling was detected radioautographically as a flux of labeled precursor molecules from a prelabeled to a recipient population of glial cells in coculture. The cocultures were secondarily incubated with [3H]thymidine to label the nuclei of S-phase recipient cells. In the cocultures there was a higher rate of nuclear labeling in coupled than in uncoupled recipient glial cells. The results suggest that growth in subconfluent retinal glial cell cultures is modulated differentially by two types of interactions which require cell contact: growth is inhibited by interaction among nonliving cell surfaces but stimulated by metabolic cooperation among living cells.

Animals↗
PubMed1989

Ouabain-sensitive Na+-K+ ATPase pumps in cultured human retinal pigment epithelium.

The expression of Na+-K+ ATPase pumps was studied in cultured human retinal pigment epithelium (RPE). Pump site number was measured by quantitation of the specific binding of [3H]ouabain to cultures of varying density. Specific binding of [3H]ouabain was time- and concentration-dependent, and was inhibited by potassium and by excess unlabeled ouabain. Estimates of pump site number based upon specific [3H]ouabain binding indicated that the number of pumps per RPE cell was maximal in sparse cultures and declined six-fold as cultures became confluent. Pump activity, determined by measurement of specific 86Rb (rubidium) uptake, was also greater in sparse than in dense cultures. Quantitation of [3H]thymidine incorporation as a measure of cell proliferation demonstrated that proliferation in RPE cultures decreased logarithmically as culture density increased. Increased pump site number in cultured RPE, therefore, correlated with increased cell proliferation and decreased culture density. We conclude that human RPE express ouabain-sensitive Na+-K+ ATPase in vitro and maximal expression is observed in sparse, proliferating cultures.

Biological Transport, Active↗