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Biomedical subjects

J M Butler

Publications and source records attributed to J M Butler.

At least 91 records · Page 5Linked to original sources
PubMed1996

Peptide nucleic acid characterization by MALDI-TOF mass spectrometry.

Peptide nucleic acids (PNAs) are a new class of DNA mimics in which the regular nucleobases of adenine, thymine, cytosine, and guanine are connected via a peptide-like backbone. PNA molecules retain the same Watson-Crick base pairing as regular oligonucleotides, with the added benefits of greater specificity and resistance to enzymatic digestion. While the use of PNAs has grown rapidly because of their potential applications in biotechnology, little work has been done on developing analytical procedures for characterizing them. We have found matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to be an effective tool for PNA analysis. PNA molecules survive the MALDI process intact and are easily ionized with almost no multiply-charged species. These features allow mixtures to be easily characterized. Traditional protein matrices (e.g., sinapinic acid,2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid) were found to be superior to DNA matrices (e.g., trihydroxy-acetophenone, 3-hydroxypicolinic acid, picolinic acid). In addition, the new DNA matrix 6-aza-2-thiothymine worked well. The ability of MALDI-TOF-MS to ascertain PNA purity and sequence information at low picomole levels will be important as greater quality control of PNA synthesis is needed (e.g., when PNAs are used as antisense or antigene drugs).

Base Sequence↗
PubMed1996

Demonstration of synergistic effects of sulfonamides and dihydrofolate reductase/thymidylate synthase inhibitors against Neospora caninum tachyzoites in cultured cells, and characterization of mutants resistant to pyrimethamine.

OBJECTIVE: To examine the efficacies of combinations of 7 sulfonamides and 5 dihydrofolate reductase/thymidylate synthase (DHFR/TS) inhibitors against tachyzoites of Neospora caninum in cultured cells. Mutant tachyzoites that were resistant to pyrimethamine were produced and examined for resistance to other DHFR/TS inhibitors. DESIGN AND PROCEDURES: After 5 days of treatment, a cell culture flask lesion-based assay was used to determine efficacies of combinations of sulfonamides and DHFR/TS inhibitors against N caninum tachyzoites and to evaluate the sensitivity of pyrimethamine-resistant mutants of N caninum to test agents. Cultured cells that were infected with the appropriate strains of N caninum and treated or not treated (controls) with test agents were examined. Mutations were induced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or by selection for growth in permissive concentration of pyrimethamine. RESULTS: Synergism was detected for combinations of pyrimethamine, ormetoprim, trimethoprim, or diaveridine with the sulfonamides. Methotrexate did not have improved efficacy when combined with sulfonamides. Two mutants were produced that were resistant to pyrimethamine. Both mutants were resistant to other DHFR/TS inhibitors. Both mutants remained resistant to pyrimethamine in the absence of continuous exposure to the agent, indicating that the induced resistance was stable. Synergism was detected for combinations of DHFR/TS inhibitors and sulfonamides against these pyrimethamine-resistant mutants. CONCLUSIONS: Combinations of suboptimal concentrations of sulfonamides with suboptimal concentrations of DHFR/TS inhibitors results in improved efficacy of the agents in a cell culture assay. Stable resistance to pyrimethamine can be induced in N caninum tachyzoites by use of chemical mutagenesis or by selection. CLINICAL RELEVANCE: In vitro evidence indicated that combination treatment, using sulfonamides and DHFR/TS inhibitors, may be effective in treating neosporosis.

Animals↗
PubMed1995

Application of dual internal standards for precise sizing of polymerase chain reaction products using capillary electrophoresis.

Capillary electrophoresis (CE) is an analytical technique which provides rapid, high resolution analysis of amplified DNA fragments produced by the polymerase chain reaction (PCR). In this study, two internal standards are used as size markers to bracket und precisely size PCR products. The technique is applied to typing PCR products from the short tandem repeat locus HUMTH01. HUMTH01 consists of five to seven major alleles in the size range of 179-203 bp, with each allele four bp apart. Using this genetic marker, a population containing 97 individuals was examined with both polyacrylamide gel electrophoresis and CE. Identical genotypes were obtained with both techniques demonstrating the reliability of CE in DNA typing applications. The DNA analysis took place in sets of 10 with a calibration of the CE being performed between each set of samples. For the 97 samples examined, the pooled standard deviation was 0.3 bp. The observed genotype frequencies determined from the sample set did not deviate significantly from Hardy-Weinberg expectations. From these CE results, we conclude that HUMTH01 PCR products can be accurately and precisely sized by capillary electrophoresis using the method described.

Base Sequence↗
PubMed1994

Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence.

In samples where the amount of DNA is limited, the polymerase chain reaction (PCR) can amplify specific regions of the DNA. A quantitative analysis of the PCR product would be desirable to ensure sufficient DNA is available for analysis. In this study, we examine the use of capillary electrophoresis (CE) with laser fluorescence detection for quantitation of PCR products. A coated open tubular capillary was used with a non-gel sieving buffer and a fluorescent intercalating dye to obtain results within 20 minutes. Using an internal standard, peak migration time was below 0.1% relative standard deviation (R.S.D.) with a peak area precision of 3% R.S.D. In comparison to quantitation by hybridization, (i.e., slot blot) and spectrophotometric analysis, capillary electrophoresis shows distinct advantages due to its ability to separate unincorporated primers and PCR byproducts from the targeted PCR product. The results demonstrate that CE can be used to monitor the quality and quantity of the PCR product.

DNA, Viral↗
PubMed1994

Purification and initial characterization of a potential plant vacuolar targeting receptor.

Clathrin-coated vesicles are known to be involved in the transport of proteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of approximately 80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum sativum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the approximately 80 kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the approximately-80 kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.

Amino Acid Sequence↗
PubMed1994

Rapid analysis of the short tandem repeat HUMTH01 by capillary electrophoresis.

Using capillary electrophoresis, we demonstrate separation and analysis of the short tandem repeat HUMTH01 in under 10 min with 3 bp resolution. Separation of the PCR products, which range in size from 179 to 203 bp, is achieved using hydroxyethyl cellulose as the separation medium and a novel single-step voltage gradient. Internal standards on either side of the alleles are used to size the PCR products with an average standard deviation of 0.5 bp. DNA typing patterns obtained with this system are compared to samples separated by polyacrylamide slab gel electrophoresis.

Cellulose↗
PubMed1989

Fine structural defects in a case of congenital microcoria.

A case of congenital miosis associated with myopia and cataract is described. Tissue obtained at iridectomy was examined with the electron microscope and compared with a specimen of normal iris. The miotic iris was found to be hypoplastic and to display differences in the cellular density of the stroma. The contractile processes of the dilator myoepithelium were conspicuously absent from the underlying stroma, and the existing myofibrils were much disarranged.

Adult↗
PubMed1988

Recent experimental studies on the blood-aqueous barrier: the anatomical basis of the response to injury.

In the mammalian eye the tissue layers which effectively separate the neural retina and the transparent refractive media from the circulating blood are generally known as the blood-retinal barrier (BRB) and blood aqueous barrier (BAB) respectively. These have been delineated by morphologically identifying the ultrastructural features which impede the passage of molecules from the blood to the working parts of the eye; physiologically, there is a certain amount of overlap in their function, as there is free diffusion of solutes between the aqueous humour, the vitreous humour and the interstitial tissue of the neural retina. Notwithstanding, it is convenient and appropriate to maintain the distinction as the more recent work has focused on the fine structural aspects of these barrier systems. The abundant literature which describes the work leading to the identification and characterisation of these barriers has been reviewed in depth with great authority by several eminent researchers, notably the late Giuseppina Raviola, and Jose Cunha-Vaz. Certain pathological conditions, trauma--including surgical manipulation, and chemical irritation of the eye can cause significant disruption of these barriers with important clinical consequences. In our laboratory interest has centred around vascular and epithelial changes which occur when the eye is injured and which can lead to a breakdown of the BAB. More recently attention has been drawn to functional and behavioural differences between species, particularly with regard to the relative stability of the barrier.

Animals↗
PubMed1985

Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye.

The occurrence, distribution, and origin of immunoreactive calcitonin gene-related peptide (CGRP) in nerves of rat, guinea pig, cat, and monkey eyes were investigated by immunocytochemistry, radioimmunoassay, and chromatography. A rich network of CGRP-immunoreactive nerve fibres was noted in the anterior uvea, which was widely distributed in both dilator and constrictor pupillae muscles and extended to the ciliary body and uveal blood vessels. Numerous CGRP-immunoreactive neuronal cells were present in the trigeminal ganglion. The extractable CGRP was 8.6 +/- 1.8 pmoles/gm of tissue in the iris and 44.0 +/- 8.1 pmoles/gm in the trigeminal ganglion. Following damage to the Gasserian ganglion a marked decrease of CGRP immunoreactivity was observed in the anterior uvea (control 11.3 +/- 1.6 pmoles/gm; operated 1.4 +/- 0.1 pmoles/gm) confirming the origin of the immunoreactive fibres from trigeminal primary sensory neurons. The sensory nature of the CGRP-immunoreactive fibres was substantiated by the depletion of CGRP immunoreactivity observed after treatment with capsaicin, which is known to cause selective degeneration of sensory neurons. Comparative studies on the distribution and colocalisation of CGRP and the putative sensory neurotransmitter substance P revealed a closely parallel distribution of the two peptides in certain regions of the uvea and their coexistence in a subpopulation of trigeminal primary sensory neurons. This study suggests that the sensory nervous system in the eye is more heterogeneous in terms of its putative neurotransmitters than previously indicated.

Animals↗
PubMed1985

Calcitonin gene-related polypeptide as a mediator of the neurogenic ocular injury response.

Calcitonin gene-related polypeptide (CGRP) has been localised immunochemically within the rat and guinea pig anterior uvea to nerve fibres of trigeminal origin. As with substance P (1-3) the level of CGRP in the iris-ciliary body is depleted after thermal damage to the Gasserian ganglion and elevated in chronically sympathectically denervated eyes. Unlike substance P, a potent pupillary constrictor (4,5), CGRP has no notable miotic action, but does, however, cause an elevation of the intraocular pressure (IOP) accompanied by disruption of the blood-aqueous barrier. It is proposed that the diverse actions of these two sensory neuropeptides conjointly mediate the antidromic ocular injury response.

Afferent Pathways↗
PubMed1985

Immunopharmacology of the atopic diseases.

The atopic conditions, atopic dermatitis, asthma, and allergic rhinitis, may arise as a result of infiltrating bone marrow-derived cells into skin or respiratory mucosae. Release of inflammatory factors from these cells could account for cutaneous vascular instability and pruritus in atopic dermatitis. Erythema and itch have been induced by experimental stress interviews and by blind food challenges. In the latter, increased plasma histamine was detected and correlated with cutaneous reactions. Basophils from patients with atopic dermatitis have increased histamine release after exposure to immunologic or nonimmunologic lectin stimuli. This increased releasability may relate to inadequate cyclic AMP regulation of cell function. We have found that leukocytes of patients with atopic dermatitis have elevated phosphodiesterase activity and consequently reduced intracellular cyclic AMP. Exposure of the cells to a phosphodiesterase inhibitor caused considerable reduction in histamine release. Similarly, exposure of atopic B lymphocytes to a phosphodiesterase inhibitor greatly reduced the high spontaneous IgE synthesis in mononuclear leukocyte cultures. Elevated leukocyte phosphodiesterase activity may also serve as a marker for the atopic diathesis. We have found elevated enzyme activity in umbilical cord blood from newborns with atopic parents, suggesting that this defect may relate to a genetically determined defect. These studies have provided insight into basic abnormalities associated with atopic dermatitis and the atopic diathesis. Defects of regulatory mechanisms in immune and inflammatory cells may help explain the seemingly disparate disorders of physiologic, pharmacologic, and immunologic systems in atopy.

Asthma↗
PubMed1985

Basophil histamine release in atopic dermatitis and its relationship to disordered cyclic nucleotide metabolism.

Maximal histamine release (HR) from leucocytes, in response to Concanavalin A (Con A) was significantly higher in a group of 16 adults with moderate to severe atopic dermatitis (AD) when compared to 13 non-atopic adults. In a further 4 adults with AD, HR was similar to that in the normals, suggesting the existence of 'high releaser' and 'low releaser' subsets within the AD group. Leucocyte cyclic AMP phosphodiesterase (PDE) activity was significantly higher in the 'high releaser' group compared to the 'low releaser' and normal groups. High and low HR responses showed strong correlations with high and low PDE. Pre-treatment of leucocytes from 'high releasers' with the experimental PDE PDE inhibitor RO-20-1724 reduced the HR to normal levels. These findings suggest that increased histamine 'releasability' in AD is related to abnormalities in cyclic nucleotide regulation. No significant HR could be demonstrated in response to a range of concentrations of methacholine in 'high releaser' atopics and normals. Methacholine also did not affect HR in response to maximal Con A stimulation in 'high releaser' atopics. Basophil percentages within the leucocyte preparation and the histamine content per basophil, were not significantly different between the atopics and normals. Con A-stimulated histamine release did not correlate significantly with serum IgE levels.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Morphological evidence for the transfer of anionic macromolecules from the interior of the eye to the blood stream.

We have either introduced into the vitreous space or perfused through the anterior chamber of macaque monkey eyes two anionic tracers, anionic ferritin (AF) and horseradish peroxidase (HRP) and a cationic probe, cationic ferritin (CF). We have observed that the anionic molecules, but not the cationic one, are transported to the blood stream by plasmalemmal vesicles of the endothelial cells in both the retinal and the iridial vasculature. We suggest that a variety of organic anions of different MW which are commonly present in the eye tissues may be returned to the blood by the same morphological mechanism.

Animals↗
PubMed1985

Asymmetric distribution of charged domains on the two fronts of the endothelium of iris blood vessels.

The authors have studied the distribution of anionic and cationic sites on both luminal and abluminal endothelial aspects of iridial vessels in Macaca mulatta and Macaca fascicularis. With the animals in general anesthesia, anionic ferritin (AF) and cationic ferritin (CF) were either injected intravenam or perfused at known intraocular pressure (15-20 mmHg) through the anterior chamber. AF introduced intravenam was retained in the vessels' lumen. The tight junctions between the endothelial cells were impermeable and the plasmalemmal vesicles did not transport tracer to the iridial stroma. In contrast, when perfused through the anterior chamber, AF was present in the vessels' lumen. Here again the tight junctions between the endothelial cells were impermeable, but AF was contained within a great number of plasmalemmal vesicles. Iridial vessels were impermeable to CF perfused into the lumen, but a continuous layer of CF particles was found to adhere to the luminal plasma membrane. When perfused through the anterior chamber, CF was bound to the proteoglycans associated with collagen fibrils of the iridial stroma and basal laminae of stromal, pericytic, and endothelial cells but was never found in the vessels' lumen. These results indicate that different electrical charges are associated with the plasmalemmal vesicles on the luminal and abluminal fronts of iridial vessels. The authors suggest that in these vessels a unidirectional vesicular transport is responsible for the selective movement of anionic organic substances from the tissues of the eye to the bloodstream.

Animals↗
PubMed1984

Effects of VIIth (facial) nerve degeneration on vasoactive intestinal polypeptide and substance P levels in ocular and orbital tissues of the rabbit.

Levels of vasoactive intestinal polypeptide (VIP)- and substance P (SP)-like immunoreactivity were measured in ocular and orbital tissues of albino rabbits. Substantial amounts of VIP were detected in the choroid (22.6 +/- 3.6 pmol g-1), and in the lacrimal (13.6 +/- 4.4 pmol g-1) and Harderian glands (20.2 +/- 4.9 pmol g-1). Somewhat less was found in the anterior uvea (3.6 +/- 1.1 pmol g-1), retina (5.4 +/- 1.3 pmol g-1) and optic nerve head (4.1 +/- 1.1 pmol g-1). Other tissues, including conjunctiva and extraocular muscle showed very little VIP-like immunoreactivity. Seven days after diathermic damage to the region of the pterygopalatine ganglion VIP was virtually eliminated from all these tissues. SP levels were also reduced, notably in the anterior uvea, probably due to concurrent destruction of sensory fibres. Electron microscopy revealed extensive degeneration of unmyelinated axons in the short ciliary nerves and in the choroid. No changes in ocular VIP levels were detected after sympathetic denervation, although a significant rise in SP was observed in the anterior uvea. The decrease in retinal VIP, believed to be confined to the amacrine cells, is considered to be a result of post-operative lid closure, rather than of VIIth nerve degeneration. Nevertheless, with this exception, VIP in ocular and orbital tissues of the rabbit appears to be contained exclusively within parasympathetic fibres of facial nerve origin.

Animals↗