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Biomedical subjects

L M Smith

Publications and source records attributed to L M Smith.

At least 217 records · Page 12Linked to original sources
PubMed1982

Amplified dihydrofolate reductase genes are located in chromosome regions containing DNA that replicates during the first half of S-phase.

To obtain a better understanding of the relationship between metaphase chromosome banding patterns and genome organization, attention was focused on regions of metaphase chromosomes that were found to contain the genes for a specific cellular enzyme, dihydrofolate reductase (DHFR). These studies involved the use of highly methotrexate-resistant mouse lymphoblastoid cells (L5178YR), which contain approximately 300 times the number of DHFR genes present in parental cells (L5178YS). Karyotypic analysis revealed the presence of two very large, nonhomologous, marker chromosomes that were absent in the parental line. In situ hybridization of 3H-labeled cloned DHFR cDNA to metaphase chromosomes of L5178YR cells was used to localize the DHFR genes to a very large Giemsa (G)-negative region on each of the two large marker chromosomes. Regional patterns of DNA replication in metaphase chromosomes were studied by autoradiographic visualization of [3H]thymidine incorporation and by fluorescent microscopic visualization of bromodeoxyuridine incorporation. Because the amplified DHFR genes were present within two prominent cytogenetic regions on two easily identifiable chromosomes, it was possible to observe the following. The amplified DHFR genes were located in chromosome regions that replicated at the same time during the first half of a 9-h-S-phase. DNA replication began simultaneously and terminated simultaneously at many locations throughout each amplified region. We conclude that transcriptionally active DHFR genes are located within large G-negative regions of metaphase chromosomes and that the DNA within these regions replicates during the first half of S-phase.

Animals↗
PubMed1982

Lipid monolayer-coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils.

We have used epifluorescence and photobleaching techniques to study the lateral distribution and motion of fluorescein-conjugated Fab fragments of anti-C3b receptor antibody bound to human neutrophils when the cells rest on various solid supports (microscope slides or cover slips). Supports composed of quartz, glass, or alkylated glass induced cellular adhesion, spreading, and an extensive lateral redistribution of C3b receptors (but not HLA antigens). The neutrophil C3b receptors become patchy, and the patches apparently undergo nonrandom translational motion. Many patches are found on the upper surfaces of the cells removed from the region of cell membrane-glass contact. In contrast, neutrophils supported by lipid monolayer-coated glass do not adhere or spread, and the C3b receptor remains uniform and diffuses freely (D approximately equal to 2 X 10(-10) cm2/s).

Antibodies, Monoclonal↗
PubMed1982

Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae.

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.

Cycloheximide↗
PubMed1981

Measurement of rotational motion in membranes using fluorescence recovery after photobleaching.

A method has been developed for the measurement of the rotational motion of membrane components. In this method fluorescent molecules whose transition dipole moments lie in a given direction are preferentially destroyed with a short intense burst of polarized laser radiation. The fluorescence intensity, excited with a low intensity observation beam of polarized laser radiation, changes with time as the remaining fluorescent molecules rotate. The feasibility of the method has been demonstrated in a study of the rotation of the fluorescent lipid probe, dil ([bis,-2-(N-octadecyl-3,3-dimethyl-1-benzo[b]pyrrole]-trimethincyanine iodide) incorporated into membranes composed of distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC) and 0.20 mol% cholesterol, below the main chain-melting transition temperatures of the phosphatidylcholines. Rotation times in the 0.6-800 s range were observed. The fluorescence recovery (or decay) curves are in satisfactory agreement with theoretical calculations.

Cholesterol↗
PubMed1981

Pattern photobleaching of fluorescent lipid vesicles using polarized laser light.

A burst of linearly polarized laser radiation incident on a spherical lipid vesicle, liposome, or biological cell can produce a well-defined nonuniform distribution of membrane-bound fluorescent molecules, provided the absorption transition dipole moment of the fluorescent label has a nonrandom orientation relative to the membrane surface and can be photobleached by the laser radiation. The return (recovery) of fluorescent membrane-bound molecules to a uniform distribution can be monitored using the same polarized radiation source. Under appropriate conditions this recovery is characterized by a single exponential time constant tau. This time constant is related to the radius R of the vesicle and the lateral diffusion coefficient D of the fluorescent membrane-bound molecules by the equation R2 = 6D tau. In the case of vesicle membranes this result is not limited by diffraction and so should be applicable to vesicles whose radii are less than the wavelength of light. The above considerations are illustrated by the polarized light photobleaching-recovery of lipid vesicles containing a fluorescent lipid, N-4-nitro-benzo-2-oxa,1,3-diazole l-alpha-dimyristoylphosphatidylethanolamine (NBD-DMPE).

Diffusion↗
PubMed1980

Lateral diffusion of M-13 coat protein in mixtures of phosphatidylcholine and cholesterol.

The translational diffusion of fluorescent-labeled M-13 phage coat protein (FITC-M-13), an integral membrane protein, has been measured in mixtures of phosphatidylcholines and cholesterol, using a pattern photobleaching technique. At temperatures below the chain-melting transition temperature of dimyristoylphosphatidylcholine (DMPC) (23.8 degrees C), the lateral diffusion coefficient of M-13 shows a marked increase when the cholesterol concentration is increased above 20 mol %. A similar marked increase in the lateral diffusion coefficient of a fluorescent phospholipid is also observed. At temperatures above the chain-melting transition temperature of DMPC, a minimum is observed in the lateral diffusion coefficient of FITC-M-13 for cholesterol concentrations in the vicinity of 25 mol %. This minimum in the diffusion coefficient of FITC-M-13 is also observed at 25 mol % cholesterol in egg phosphatidylcholine at 15 degrees C. No such minimum is observed for the lateral diffusion coefficient of the fluorescent lipid. The lateral diffusion coefficient of FITC-M-13 is large (greater than 10(-9) cm2/s) at all cholesterol concentrations for temperatures above the chain-melting transition temperature of the phosphatidylcholine. Several other proteins contain hydrophobic regions similar to that of the M-13 coat protein. We anticipate that a number of proteins of this type will show similar diffusional behavior, in particular exhibiting rapid diffusion throughout a wide range of lipid composition.

Cholesterol↗
PubMed1980

The thalamocortical projection in Pseudemys turtles: a quantitative electron microscopic study.

Thalamic fibers in the cortex of Pseudemys turtles were studied with the electron microscope to determine the type of synaptic vesicle they contain, the type of membrane differentiation they form, and the type of processes they contact. Following unilateral removal of the thalamus, all degenerating thalamic axon terminals are located in the outer third of the molecular layer in the rostral half of general cortex. In the middle of this zone they constitute as much as 25% of all vesicle-containing profiles. The degenerated terminals appear as electron opaque profiles, most commonly with a uniform opacity. They contain round agranular vesicles and form synapses with asymmetrical membrane differentiations. They synapse mainly on dendritic spines containing mitochondria and/or membranous sacs, although some thalamic fibers contact small clear spines, dendrites, and, rarely, cell bodies. Counts show that 86% of degenerated contacts are on dendritic spines and 14% on dendritic shafts. The spines probably all belong to the dendrites of the pyramidal cells, whose somata are located in the deep cellular layer. The dendritic shafts and somata are most likely those of the aspinous stellate neurons located in the molecular layer. Although these stellate cells are not sufficiently numerous to form a cell "layer," each transverse section through thalamic recipient cortex contains about nine of these cells and they occur in a ratio of 1:37 to pyramidal cells in the underlying main cell layer. We have calculated that in a rectangular solid of turtle cortex whose dimensions are 1 mm X 1 mm X the depth from pial surface to the underlying ventricle, there are 5.2 million thalamic fiber contacts (all in the outer 100 micrometers), 15,000 pyramidal neurons in the main cell layer, and 400 stellate cells in the molecular layer. Of the 5.2 million thalamic synapses, 0.7 million contact stellate cells and 4.5 million contact pyramidal cells. Thus each stellate cell in the molecular layer receives on the average 1,800 thalamic fiber contacts, while each pyramidal cell receives only 300 thalamic fiber synapses on the distal portion of its dendrites. The calculations lead to the conclusion that individual stellate cells receive at least six times more thalamic fiber synapses than individual pyramidal cells in turtle cortex. We suggest that the stellate cells in the thalamic input zone are inhibitory and that each thalamic volley not only excites efferent pyramidal cells but is also a powerful activator of inhibitory interneurons.

Afferent Pathways↗
PubMed1980

Syntheses and evaluation of ketals, hemithioketals, and dithioketals of conjugated styryl ketones principally for antineoplastic activity.

Ketals, hemithioketals, and dithioketals of nuclear-substituted styryl ketones were prepared as latentiated forms of the ketones. This undertaking was based on the premise that there is increased acidity in tumors compared to normal tissue, and thus preferential regeneration of the ketone in neoplastic tissue may occur. Attempts to form 1,3-dioxolans of Mannich bases were unsuccessful. The prepared compounds did not possess significant anticancer properties, but analgesic, antiinflammatory, antihistaminic, and antimicrobial activities were found in the prepared Mannich bases.

Analgesics↗
PubMed1980

Lateral distribution and diffusion of the C3b receptor of complement, HLA antigens, and lipid probes in peripheral blood leukocytes.

Fluorescence microscopy and fluorescence redistribution after pattern photobleaching have been used to measure the distribution and motion of a number of fluorescent molecules bound to the plasma membranes of human leukocytes. The fluorescent molecules include fluorescein-labeled F(ab')2 and Fab' fragments of an anti-C3b receptor antibody, fluorescein-labeled IgG and Fab fragments of a monoclonal anti-HLA antibody, and the two lipid probes 3,3'-dioctadecylindocyanine and N-4-nitrobenzo-2-oxa-1,3-diazole L-alpha-dimyristoyl phosphatidylethanolamine. From these studies we have concluded that the C3b receptors on human polymorphonuclear leukocytes and monocytes are predominantly present in discrete clusters. No diffusive motion of these clusters could be detected. In contrast, HLA antigens hve a dispersed distribution and have diffusion coefficients between 10(-9) and 10(-10) cm2/sec in these peripheral blood leukocytes. Cell-membrane-bound monoclonal IgG anti-HLA undergoes a rapid patching in all these leukocytes except lymphocytes. In about 50% of the lymphocytes, fluoresceinated IgG diffuses slowly, if at all, whereas in the other 50%, fluoresceinated IgG diffuses with a diffusion coefficient of 6.9 X 10(-10) cm2/sec. The two above-mentioned fluorescent lipid probes have the usual rapid lipid diffusion coefficient (approximately 10(-8) cm2/sec) in leukocytes.

Antigen-Antibody Reactions↗
PubMed1980

Negative chemical ionization studied of human and food chain contamination with xenobiotic chemicals.

Negative chemical ionization mass spectrometry with a mixture of isobutane, methylene chloride, and oxygen as the reagent gas has been used to explore contamination of environmental substrates with xenobiotic chemicals. The substrates in question, fish tissue, human seminal plasma, and human adipose tissue, were cleaned up by one of the following three cleanup procedures: (1) continuous liquid-liquid extraction steam distillation; (2) gel-permeation chromatography; and (3) adsorption on activated carbon followed by elution with toluene. The third procedure was used only for the examination of planar polychlorinated aromatic hydrocarbons in environmental samples. Using these techniques, we have found evidence for contamination of fish samples with polychloronaphthalenes, polychlorostyrenes, polychlorobiphenyls, polychlorodibenzofurans, and polychlorodibenzodioxins among other chemicals. The polychlorodibenzodioxins appeared only in the spectra of extracts of fish obtained from the Tittabawassee River at Midland Michigan. The polychlorodibenzofuran ions appeared in NCI mass spectra of fish that were significantly contaminated (above 2 ppm) with polychlorobiphenyls. Toxic substances occurring in human seminal plasma included pentachlorophenol, hexachlorobenzene, DDT metabolites, and polychlorobiphenyls. We have investigated toxic substances in human seminal plasma because of the apparent decrease in sperm density in U.S. males over the last 30 years. Results of screening human adipose tissue for contamination with xenobiotic chemicals have been largely coincident with result of the EPA human monitoring program. Polychlorobiphenyls, DDT metabolites, nonachlor, and chlordane have appeared in most samples examined. Detection limits for all of these chemicals were of the order of 1 ppb.

Adipose Tissue↗
PubMed1979

Lateral diffusion of M-13 coat protein in model membranes.

A fluorescent derivative of the M-13 phage coat protein (molecular weight 5260) was reconsituted into oriented multilayers and giant liposomes of dimyristoylphosphatidylcholine. The rate of lateral diffusion of the labeled protein in the fluid phase was measured as a function of temperature and found to be comparable to that of lipid probes. The protein was found to have a nonuniform lateral distribution in the solid phase of both types of model membranes. Cardiolipin (0.5 mol %) included in the multibilayers did not have any substantial effect upon the rate of diffusion.

Coliphages↗
PubMed1979

Antibodies bound to lipid haptens in model membranes diffuse as rapidly as the lipids themselves.

A pattern photobleaching method has been used to measure the rates of lateral diffusion of fluorescent-labeled specific anti-nitroxide IgG bound with both combining sites to nitroxide-containing phospholipids in liposomal membranes composed of dimyristoyl phosphatidylcholine at 28 degrees C ("fluid"), dipalmitoyl phosphatidylcholine at 32 degrees C ("Solid"), and dipalmitoyl phosphatidylcholine containing 15 or 25 mol% cholesterol ("solid" or "fluid," respectively, at 32 degrees C). The diffusion coefficients of the bound immunoglobulin were found to be the same as those of fluorescent-labeled phospholipids in each case even though these diffusion coefficients range from 10(-11) to 10(-8) cm2/sec. Hapten-containing liposomal membranes of the type studied here have previously been shown to elicit a number of antibody-dependent immune responses. Therefore, this work indicates that membrane-bound but otherwise freely diffusing antibodies are sufficient for these reponses.

Antigen-Antibody Complex↗
PubMed1978

Potential hypocholesteremic derivatives of styrylacetic acid II: cis- and trans-3-methyl-4-phenyl-3-butenoic acid analogs.

The synthesis and preliminary biological testing for in vitro cholesterol biosynthesis inhibitory activity of 2-indeneacetic acid, 2-methyl-1,2-dihydro-2-naphthoic acid, and their 5- and 7-chloro derivatives, respectively are described. These compounds were prepared as trans- and cis-analogs of the known antilipemic agent 3-methyl-4-phenyl-3-butenoic acid. Although both series of compounds showed cholesterol biosynthesis inhibitory properties, chloro substitution enhanced potency only in the cis-system. These findings are discussed in terms of a possible relationship between the cis-compounds and clofibrate-type antilipemic agents.

Acetates↗
PubMed1978

Determination of Indole-3-acetic Acid in Douglas Fir Using a Deuterated Analog and Selected Ion Monitoring: Comparison of Microquantities in Seedling and Adult Tree.

Indole-3-acetic acid (IAA) content in shoot tips of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) trees and seedlings was determined by combined gas chromatography-mass spectrometry using a deuterated analog (d(2)-IAA) as an internal standard and the technique of selected ion monitoring. Ratios of the peak heights of the deuterated analog internal standard to endogenous IAA revealed a slightly higher content of IAA in seedlings compared with the shoot tips collected in June. The relatively high level of IAA (2.9 micrograms per gram fresh weight) in seedlings is discussed in relation to in vitro propagation of this species.

Journal Article↗