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Biomedical subjects

J McKeon

Publications and source records attributed to J McKeon.

At least 19 recordsLinked to original sources
PubMed2001

Quantitation of intracellular concentration of a delivered morpholino oligomer by capillary electrophoresis-laser- induced fluorescence: correlation with upregulation of luciferase gene expression.

Antisense oligonucleotides have shown great promise over the past several years as viable drugs to combat various forms of cancer and viral diseases. However, quantitative detection to monitor cellular association is difficult using conventional methods such as radiolabeling of the oligonucleotide or fluorescence confocal microscopy. In this paper quantitation of intracellular concentration of the morpholino oligonucleotide is investigated using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). HeLa cells, which produce luciferase as the antisense oligomer enters the cell, were scrape-loaded with varying concentrations of the morpholino antisense. The intracellular antisense concentration measured by CE-LIF was found to correlate with those obtained with the cellular functional assay based on upregulation of luciferase. Intracellular concentrations of the antisense were found to be in the range of 6 to 29 nmol/g total cell protein, depending on the amounts that were scrape-loaded. To our best knowledge, this is the first reported quantitative correlation between delivered antisense concentration in a cell extract and the subsequent antisense upregulation of gene expression.

Cell Membrane↗
PubMed2001

Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection.

We demonstrate the use of simple extraction procedures to separate nuclear and cytoplasmic material from cell extracts, which have been scrape-loaded with a 2-O-methyl phosphorothioate antisense oligonucleotide. Separation and quantitation of the fluorescein-labeled antisense and the flourescein isothiocyanate (FITC)-dextran (molecular weight 40000) as an internal standard is done using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). The bulky FITC-dextran is unable to penetrate the nuclear membrane thereby making it a quantitative indicator of any overlap between the nuclear and cytoplasmic materials during separation of the two phases. Using this procedure, the fluorescein-labeled phosphorothioate oligomer was quantitated at 4.1 x 10(-13) and 3.4x 10(-14) mol antisense/microg-total cellular protein in the nuclear and cytoplasmic extracts respectively following scrape-load delivery of the phosphorothioate to a batch of confluent HeLa cells at a concentration of 0.5 microM (5 x 10(-10) total moles of oligomer). Additionally, gene expression was monitored by measurement of the luciferase reporter protein activity. Scrape-load, spontaneous and liposomal delivery were investigated and compared for subcellular distribution of the oligomer and subsequent gene expression.

Cell Fractionation↗
PubMed1995

Avoidance and management of adverse reactions to antituberculosis drugs.

Potent antibiotics are required to cure tuberculosis and reduce the burden of illness in the community. Minor adverse effects are commonly encountered and can be managed by reassurance and explanation. Significant hypersensitivity reactions require cessation of all antituberculosis drugs. Adverse effects should be treated appropriately. An effective antituberculosis regimen should be reestablished as soon as possible. Desensitisation may be necessary if suitable alternative drugs cannot be used. In the event of drug-induced hepatitis, all hepatotoxic drugs should be ceased until symptoms resolve and liver function tests return to normal. Other significant direct toxic effects should be promptly detected and appropriately treated. A thorough knowledge of potential adverse reactions and pharmacokinetics is essential for any physician using antituberculosis drugs.

Aging↗
PubMed1994

Mutations in some Polycomb group genes of Drosophila interfere with regulation of segmentation genes.

Mutations in several Polycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting that Pc group genes may regulate the segmentation genes of Drosophila. We show that individuals doubly heterozygous for mutations in polyhomeotic and six other Pc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations of Pc group and segmentation mutations for enhancement of adult and embryonic segmentation defects. Posterior sex combs and polyhomeotic interact with Krüppel and enhance embryonic phenotypes of hunchback and knirps, and polyhomeotic enhances even-skipped. Surprisingly, flies carrying duplications of extra sex combs (esc), that were heterozygous for mutations of even-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression of eve is suppressed by duplications of esc. However, esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated with Pc group genes. Duplications of nine other Pc group genes have no detectable effect on eve. Expression of engrailed (en) was abnormal in the central nervous systems of most Pc group mutants. These results support a role for Pc genes in regulation of some segmentation genes, and suggest that esc may act differently from other Pc group genes.

Abdomen↗
PubMed1992

Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor in Drosophila.

Chromatin footprinting in Drosophila tissue culture cells has detected the binding of a non-histone protein at +8 of the distal Adh RNA start site, on a 10-bp direct repeat motif abutting a nucleosome positioned over the inactive Adh distal promoter. Alternatively the active promoter is bound by a transcription initiation complex. We have characterized and purified a protein Adf-2 that binds specifically to this direct repeat motif 5'TCTCAGTGCA3', present at +8 and -202 of the distal RNA start site. DNase I footprinting, methylation interference, and UV-crosslinking analyses showed that both direct repeats interact in vitro with a nuclear protein of approximately 120 kilodaltons (kDa). We purified Adf-2 through multiple rounds of sequence-specific DNA affinity chromatography. Southwestern analysis showed that the purified 120 KDa polypeptide binds the Adf-2 motif efficiently as a monomer or homomultimer. In vivo titrations of Adf-2 activity with the Adf-2 motif by transient co-transfection competitions in different Drosophila cell lines suggested that Adf-2 is a cell-specific repressor. Adf-2 has been detected ubiquitously in vitro, but is functional in vivo as a sequence-specific DNA binding protein and repressor only in the cells that have the inactive distal promoter. We discuss the possibility that an activation process is required for Adf-2 protein to bind DNA and function in vivo.

Alcohol Dehydrogenase↗
PubMed1991

Orthopedic surgery and rehabilitation for the prolongation of brace-free ambulation of patients with Duchenne muscular dystrophy.

The purpose of this study was to prospectively evaluate the results of a short comprehensive program involving early lower extremity musculotendinous surgery followed by a definitive course of rehabilitation on contractures and the duration of ambulation for patients with Duchenne muscular dystrophy. Seven patients were treated while ambulating with little difficulty and six were treated just before or after becoming wheelchair-dependent. Predicted post-treatment duration of ambulation was calculated from established clinical criteria. Actual prolongation of brace-free ambulation after treatment was a mean of 0.8 yr greater than predicted for the group as a whole but 0.93 yr for the group treated early by comparison with 0.63 yr for those treated according to the customary approach. The number of falls significantly decreased from 84 +/- 87 to 1 +/- 1 per month postoperatively (P less than 0.05); however, the speed of ambulation over a distance of 10 yards decreased from 10.2 +/- 4 s to 12.1 +/- 7.3 s. Three patients who had tibialis posterior transfers retained antigravity plus dorsiflexor strength and continue to wear normal footwear 2.5, 3.7 and 4.0 yr after loss of ambulation. We conclude that ambulation becomes more stable and brace-free ambulation may be prolonged by a comprehensive program of early orthopedic surgery and rehabilitation. Earlier intervention is also better tolerated.

Adolescent↗
PubMed1989

Increased expression of the gene for the pro alpha 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta.

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.

Basement Membrane↗
PubMed1987

Roles of cis-acting elements and chromatin structure in Drosophila alcohol dehydrogenase gene expression.

The alcohol dehydrogenase (Adh) gene of D. melanogaster is transcribed from two different promoters during fly development: the distal (adult) and the proximal (embryonic-larval). Certain aspects of Adh gene regulation are represented in Drosophila continuous cell lines. We have used Drosophila tissue culture cells in an in vivo transient expression assay to delimit cis-acting sequences affecting Adh expression, and to investigate the role of chromatin structure in Adh gene regulation. These studies show that positive cis-acting elements of the distal promoter can exist in at least 2 alternative chromatin configurations. There is a close correlation between specific transcriptional activity of the Adh distal promoter and a defined, localized chromatin structural change that indicates altered DNA-protein interactions. Thus, chromatin structure appears to play a role in regulating the accessibility of defined positive cis-acting regulatory sequences of Adh to transcription factors and the transcription machinery.

Alcohol Dehydrogenase↗
PubMed1987

Gap junction assembly in the preimplantation mouse conceptus is independent of microtubules, microfilaments, cell flattening, and cytokinesis.

Gap junctions first appear during compaction in the eight-cell stage of mouse development. Their assembly can be initiated in the near absence of transcription and protein synthesis from the four-cell stage, indicating the existence of preformed precursors. We have investigated the temporal control of this event, focusing on the possible involvement of the cytoskeleton, cell flattening, and cytokinesis. Embryos in various cleavage stages were treated with cytochalasins, to disrupt microfilaments and block cell flattening, cytokinesis, or both, or nocodazole, to promote microtubule depolymerization. To assess their capacity to initiate gap junction assembly after such treatments, the embryos were then aggregated with communication-competent, compacted embryos that had been labeled with carboxyfluorescein diacetate. Passage of the fluorescent dye, carboxyfluorescein, from labeled to unlabeled embryo was taken as evidence that interembryonic junction formation had occurred. The capacity to assemble gap junctions was acquired at the normal time by embryos prevented by cytochalasin treatment from undergoing cell flattening or any cytokinesis from fertilization onward. Likewise, treatment with nocodazole beginning in the four-cell or early eight-cell stage did not interfere with gap junction assembly. Neither drug affected the inability of four-cell embryos to assemble gap junctions prematurely. We conclude that intact microfilament or microtubule networks are not required for gap junction assembly in this system, nor do they restrain junctional precursors from assembling prematurely. Furthermore, the timing of gap junction assembly is not linked to cell flattening, cytokinesis, or cell number.

Actin Cytoskeleton↗
PubMed1986

Defects in the processing of procollagen to collagen are demonstrable in cultured fibroblasts from patients with the Ehlers-Danlos and osteogenesis imperfecta syndromes.

This is a study of the processing of procollagen to collagen in cultures of skin and tendon fibroblasts. Processing was markedly increased by growing cells for 2-4 days postconfluence and then adding ascorbate to the medium for 2 days prior to labeling with [3H] proline. With this system, more than two-thirds of the pro-alpha chains of type I procollagen in the culture medium, and more than 90% of those in the cell layer, were rapidly processed to pC-alpha, pN-alpha, or alpha chains. Purified, exogenous procollagen was also rapidly processed in cell-free culture medium. The results showed for the first time that exogenous procollagen can be processed in conditioned cell-free medium. The system was then used to compare the processing of procollagen in the medium of normal fibroblasts, cells from one bovine and four human variants of osteogenesis imperfecta, and those from eight human variants of the Ehlers-Danlos syndrome. The cells could be divided into three groups, based on their ability to process type I procollagen: normal, consistently slow, and very slow. The cause of the decreased processing was shown to be associated with either a mutation causing a shortening of an alpha chain or decreased activity of procollagen N-proteinase in cell-free culture medium. Decreased processing of procollagen to collagen occurred with cultured fibroblasts from patients with different forms of both osteogenesis imperfecta and Ehlers-Danlos syndrome. Both of these disease syndromes are associated with abnormalities in the structure or metabolism of procollagen in fibrous connective tissues, bones, and teeth. The results show that defects in the structure, synthesis, or processing of procollagen are readily demonstrated with cultured fibroblasts.

Adolescent↗
PubMed1984

Life events and personality traits in obsessive-compulsive neurosis.

Twenty-five patients with obsessive-compulsive neurosis and matched controls had their life event scores (Paykel's Life Event Schedule) rated for the year prior to the onset of illness and the date of interview, respectively. The Standard Assessment of Personality Schedule, whose high inter-temporal and inter-informant reliability was confirmed, was used to rate the patients' premorbid personality. The obsessive-compulsive patients' mean life event score was significantly higher than the control subjects; and this excess spanned the six months prior to the onset of illness. Patients with abnormal personality traits (obsessional, anxious and self-conscious) experienced significantly fewer life events than those without such traits.

Humans↗