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Biomedical subjects

R A Lewis

Publications and source records attributed to R A Lewis.

At least 235 records · Page 13Linked to original sources
PubMed1987

Deoxyadenosine deamination and phosphorylation in rat liver mitochondria.

1. Deoxyadenosine metabolism was measured in freshly isolated mitochondria; these organelles took up the deoxynucleoside and formed three detectable products: deoxyinosine, dAMP and dIMP. 2. Enzyme extracts prepared from sonicated mitochondria exhibited deoxyadenosine deaminase, deoxyadenosine kinase, dAMP deaminase and deoxyinosine kinase activities. 3. These data suggest that deoxyadenosine was initially altered in mitochondria by at least two metabolic reactions--deamination and phosphorylation. Deoxyinosine and dAMP were produced. 4. These two products were subsequently phosphorylated and deaminated, respectively to produce dIMP.

Adenosine Deaminase↗
PubMed1987

Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation.

Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. We have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion in Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmed by high-resolution cytogenetic analysis. We used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.

Chromosome Mapping↗
PubMed1987

Mapping the Lowe oculocerebrorenal syndrome to Xq24-q26 by use of restriction fragment length polymorphisms.

A molecular linkage analysis of four large families with the Lowe oculocerebrorenal syndrome (LS) provided a subregional localization of LS to the distal long arm of the X chromosome at Xq24-q26. Probes from two loci that identify restriction fragment length polymorphisms (RFLPs) and map to Xq24-q26 showed no recombination with LS. A maximum likelihood recombination distance (theta) = 0.00 was obtained for DXS10 with the logarithm of the odds (lod) of 6.450. For DXS42, theta = 0.00 with a lod of 5.087. Assignment of the gene or genes for LS to Xq24-q26 has the potential of improving carrier detection and providing prenatal diagnosis in families at risk for the disease.

Chromosome Mapping↗
PubMed1987

U-54494A: a unique anticonvulsant related to kappa opioid agonists.

The benzamide U-54494A was compared to U-50488H (a structurally related kappa opioid agonist), phenytoin and phenobarbital in a variety of tests of anticonvulsant and sedative activities. In electroshock convulsion antagonism studies in mice and rats, U-54494A was generally similar to the standards in regard to milligrams of potency, threshold elevation, p.o. activity and duration of action. In contrast to phenytoin and phenobarbital, both U-54494A and U-50488H were effective antagonists of the convulsions induced by the excitatory amino acid agonists (kainic, N-methyl-aspartic and quisqualic acids) and the Ca++ channel agonist (Bay K 8644). They were not, however, effective antagonists of the gamma-aminobutyric acid-related convulsants (bicuculline and pentylenetetrazole), but all four blocked audiogenic convulsions in genetically epileptic mice. U-54494A in contrast to U-50488H lacks the kappa receptor-mediated sedative and analgesic activities but the anticonvulsant properties of both compounds are antagonized by high doses of naltrexone. Further investigations of the mechanism of action of these compounds revealed that both caused a dose-related suppression of post-tetanic repetitive discharge in cats soleus nerve-muscle preparations as measured by an abolition of the obligatory potentiation of soleus muscle contractile tension. On a biochemical level, both U-54494A and U-50488H attenuate the depolarization induced uptake of 45Ca++ into forebrain synaptosomes and block the enhancement of [3H]kainic acid binding induced by CaCl2. Together these results suggest that U-54494A is a unique and selective anticonvulsant agent acting by a Ca++-related mechanism possibly through a subclass of kappa receptors.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1987

Evidence for the combined involvement of serotonergic and alpha 2 adrenergic mechanisms in the analgesic activity of tazadolene succinate.

The analgesic activity of tazadolene succinate was assessed in mice and rats subsequent to pretreatment with various depletors and antagonists of monoamine neurotransmitters. In the mouse warm plate assay, reserpine was confirmed to cause a profound antagonism of tazadolene analgesia. However, yohimbine, Sch 23390 and haloperidol, antagonists of alpha 2 adrenergic, D1 dopaminergic and D2 dopaminergic receptors respectively, did not block tazadolene, nor did the serotonin depletor, p-chlorophenylalanine. But mice treated with both the depletor and yohimbine were significantly resistant to the analgesic effects of tazadolene. Combined treatments of haloperidol or prazosin (an alpha1 adrenergic antagonist) and p-chlorophenylalanine were not similarly effective. In the rat, reserpine blocked tazadolene analgesia in both the hot plate and air induced writhing assays. Likewise, combined treatment with yohimbine and p-chlorophenylalanine also antagonized the analgesia. These results confirm the monoamine dependence of tazadolene analgesia and indicate that both serotonergic and alpha 2 adrenergic mechanisms are involved. The results further suggest that these systems may be in parallel and integrated such that either one is capable of the primary expression of the analgesic effect of tazadolene. Thus, the unique analgesia properties of tazadolene are apparently due to the ability of this compound to activate both serotonergic and alpha 2 adrenergic antinociceptive systems.

Analgesia↗
PubMed1987

Multipoint linkage analysis of loci in the proximal long arm of the human X chromosome: application to mapping the choroideremia locus.

Choroideremia (McK30310), an X-linked retinal dystrophy, causes progressive night blindness, visual field constriction, and eventual central blindness in affected males by the third to fourth decade of life. The biochemical basis of the disease is unknown, and prenatal diagnosis is not available. Subregional localization of the choroideremia locus to Xq13-22 was accomplished initially by linkage to two restriction-fragment-length polymorphisms (RFLPs), DXYS1 (Xq13-q21.1) and DXS3 (Xq21.3-22). We have now extended our linkage analysis to 12 families using nine RFLP markers between Xp11.3 and Xq26. Recombination frequencies of 0%-4% were found between choroideremia and five markers (PGK, DXS3, DXYS12, DXS72, and DXYS1) located in Xq13-22. The families were also used to measure recombination frequencies between RFLP loci to provide parameters for the program LINKMAP. Multipoint analysis with LINKMAP provided overwhelming evidence for placing the choroideremia locus within the region bounded by DXS1 (Xq11-13) and DXS17 (Xq21.3-q22). At a finer level of resolution, multipoint analysis suggested that the choroideremia locus was proximal to DXS3 (384:1 odds) rather than distal to it. Data were insufficient, however, to distinguish between a gene order that puts choroideremia between DXS3 and DXYS1 and one that places choroideremia proximal to both RFLP loci. These results provide linkage mapping of choroideremia and RFLP loci in this region that will be of use for further genetic studies as well as for clinical applications in this and other human diseases.

Choroid↗
PubMed1987

The mechanism of vascular leakage induced by leukotriene E4. Endothelial contraction.

This study identifies the microvascular target of leukotriene E4 (LTE4) by vascular labeling with carbon black and establishes the mechanism of its action at the cellular level by electron microscopy. LTE4 and its tripeptide precursor, leukotriene C4 (LTC4) were injected subcutaneously in guinea pigs. With LTE4, venular labeling was intense at 1000 and 100 ng and slight at 10 ng, with extinction at 1 ng. LTC4 induced a ring of labeled venules around a blank central area, suggestive of vasospasm. The nonpeptidyl leukotriene LTB4 induced no labeling. Histamine (1000 ng) induced an area of vascular labeling about equal to that by 1000 ng LTE4, but the labeling of individual venules was more intense. By electron microscopy, LTE4 was found to induce gaps in the endothelium of the venules; the endothelial cells adjacent to the gaps bulged into the lumen and showed wrinkled nuclei, consistent with cellular contraction. This ultrastructural evidence suggests that LTE4 increases vascular permeability by contraction of endothelial cells selectively, in the postcapillary venules, as was previously demonstrated for other inflammatory mediators, including histamine, serotonin, and bradykinin.

Animals↗
PubMed1987

Absorption and disposition kinetics of cromolyn sodium and the influence of inhalation technique.

Plasma concentrations of cromolyn sodium (SCG) have been measured in 13 normal subjects on three occasions after inhalation from a Spinhaler (20 mg) delivery system under conditions of controlled inspiratory flow rate. High inspiratory flow rates were associated with high peak plasma concentrations and areas under the plasma concentration-time curve. A 10-sec breath hold at the end of inspiration did not alter significantly the plasma concentration-time curve. Instillation of SCG (1 mg) directly into a second-order bronchus of 14 patients undergoing diagnostic examination gave plasma concentration-time curves similar to those seen in normal volunteers at high inspiratory flow rates. Additional studies in normal volunteers showed that SCG was poorly absorbed from the gastrointestinal tract. The shorter terminal half-life seen after i.v. infusion compared with inhalation indicates that the drug shows absorption rate limited disposition kinetics after inhalation. These studies in large numbers of subjects support the conclusions of limited earlier investigations that SCG is absorbed slowly but almost completely from the airways, that there is little gastrointestinal absorption of SCG, that only a small proportion of the Spinhaler dose (about 10%) reaches the airways and that the amount reaching the airways is related directly to inspiratory flow rate.

Administration, Inhalation↗
PubMed1987

DNA linkage studies of degenerative retinal diseases.

DNA linkage studies of human genetic diseases have led to rapid characterization of a number of otherwise intractable disease loci. Detection of a linked DNA marker, the first step in "reverse genetics", has permitted cloning of the genes for Duchenne muscular dystrophy, retinoblastoma and chronic granulomatosis disease, among others. Thus, the case for applying these techniques to retinitis pigmentosa and related diseases, and the urgency in capitalizing on molecular developments, is justified and compelling. The first major success regarding RP was in demonstrating linkage of the DNA marker DXS7 (L1.28) to XRP. For autosomal forms of the disease, conventional linkage studies have provided tentative evidence for linkage of ADRP to the Rh blood group on chromosome lp and for linkage of Usher's syndrome to Gc and 4q. These provisional assignments are, at least, an important starting point for DNA analysis. The Support Program for DNA Linkage Studies of Degenerative Retinal Diseases was established to provide access for the scientific community to appropriate families, using the resources of the Human Genetic Mutant Cell Repository to prepare, store and distribute lymphoblast lines. To date, two extensive, well-characterized families are included in the program: the autosomal dominant RP family UCLA-RP01, and the Usher's syndrome families LSU-US01. It is highly likely that rapid progress will be made in mapping and characterizing the inherited retinal dystrophies. We believe the support program will facilitate this progress.

Choroid↗
PubMed1987

Ocular axial length in unilateral congenital cataracts and blepharoptosis.

Biometry performed in patients with unilateral dense congenital cataracts and unilateral complete blepharoptosis did not show a consistent elongation of the involved eye. The antero-posterior axis of the visually deprived eye was longer than that in the normal fellow eye in some but shorter or unchanged in other patients. These findings are in accord with the hypothesis gained from monkey experiments that visual deprivation during infancy may deregulate axial growth of the eye. However, unlike in monkeys where axial elongation usually prevails, this effect of visual deprivation is less predictable in humans.

Adolescent↗
PubMed1986

Subcellular distribution of leukotriene C4 binding units in cultured bovine aortic endothelial cells.

The subcellular distribution of specific binding sites for [3H]leukotriene C4 ([3H]LTC4) was analyzed after sedimentation of organelles from disrupted bovine aortic endothelial cells on sucrose density gradients and was shown to be in membrane fractions I (20% sucrose) and IV (35% sucrose). Saturation binding studies of [3H]LTC4 on endothelial cell monolayers at 4 degrees C demonstrated high-affinity binding sites with a dissociation constant (Kd) of 6.8 +/- 2.2 nM (mean +/- SD) and a density of 0.12 +/- 0.02 pmol/10(6) cells. At 4 degrees C, the specific binding of [3H]LTC4 by each of the subcellular fractions reached equilibrium at 30 min and remained stable for an additional 60 min. After 30 min of incubation with [3H]LTC4, the addition of excess unlabeled LTC4 to each subcellular fraction reversed more than 70% of [3H]LTC4 binding in 10 min. The [3H]LTC4 binding activities of subcellular fractions were enhanced approximately twofold to fourfold in the presence of Ca2+, Mg2+, and Mn2+, whereas Na+, K+, and Li+ were without effect. As measured by saturation experiments, the Kd and density of LTC4 binding sites in fraction I were 4.8 +/- 1.6 nM and 16.5 +/- 1.9 pmol/mg of protein, respectively, and in fraction IV were 4.7 +/- 1.5 nM and 81.4 +/- 19 pmol/mg of protein, respectively. Inhibition of [3H]LTC4 binding in membrane-enriched subcellular fractions I and IV by LTC4 occurred with molar inhibition constant (Ki) values of 4.5 +/- 0.1 nM and 4.7 +/- 1.2 nM, respectively, whereas Ki values for LTD4 were 570 +/- 330 nM and 62.5 +/- 32.8 nM, respectively, and for LTE4 were greater than 1000 nM for each fraction; LTB4 and reduced glutathione were even less active. FPL55712, a putative antagonist of the sulfidopeptide LT components of slow reacting substance of anaphylaxis, had Ki values of 1520 +/- 800 nM and 1180 +/- 720 nM for [3H]LTC4 binding sites on membrane-enriched subcellular fractions I and IV, respectively. Thus as defined by Kd, Ki, and specificity, the LTC4 binding units that are distributed to the plasma membrane and the binding units in the subcellular fraction of greater density were similar to each other. Pretreatment of the isolated subcellular membrane fractions with trypsin abolished [3H]LTC4 binding by fraction I, enriched for the plasma membrane marker 5' nucleotidase, and that by fraction IV, enriched for the mitochondrial membrane marker succinate-cytochrome C reductase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase.

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.

Affinity Labels↗
PubMed1986

Alteration of the exonuclease activities of DNA polymerase I by captan.

DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities. Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities. When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA. However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. Inhibition and enhancement each showed an ED50 of the same value (approx. 100 microM). By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease. Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity. Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate. Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.

Bacterial Proteins↗
PubMed1986

Differential effects of captan on DNA polymerase and ribonuclease H activities of avian myeloblastosis virus reverse transcriptase.

Captan was used as an inhibitor of avian myeloblastosis virus reverse transcriptase to study the polymerase and RNase H catalytic activities. With purified enzyme, RNase H activity was 10-fold more sensitive to captan than was either the DNA-dependent or RNA-dependent DNA polymerase activity. Inhibition of the RNA-dependent polymerase activity could be prevented by dTTP. Conversely, inhibition of this polymerase activity was enhanced by template/primer. The calculated KdTTP of the uninhibited reaction was 5.6 microM. Kinetic studies allow for the proposition of a model for the interaction of captan with the polymerase active center. RNase H activity showed a sigmoidal relationship between activity and substrate concentration. Nuclease activity decreased in Vmax with no change in the Hill coefficient in the presence of captan. Addition of dithiothreitol to the incubation cocktail prevented inhibition by captan of both RNA-dependent polymerase and RNase H activities, suggesting that the (trichloromethyl)thio moiety of captan is involved in the inhibitory action. Captan inhibition suggests the presence of essential amino residues in both polymerase and RNase H active centers.

Avian Leukosis Virus↗