Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 1973"
TL;DR: A method is presented by which the gene diversity (heterozygosity) of a subdivided population can be analyzed into its components, i.e., the gene diversities within and between subpopulations.
Abstract: A method is presented by which the gene diversity (heterozygosity) of a subdivided population can be analyzed into its components, i.e., the gene diversities within and between subpopulations. This method is applicable to any population without regard to the number of alleles per locus, the pattern of evolutionary forces such as mutation, selection, and migration, and the reproductive method of the organism used. Measures of the absolute and relative magnitudes of gene differentiation among subpopulations are also proposed.
8,465 citations
TL;DR: It is proposed that a ring system sufficiently planar for a stacking interaction with DNA base pairs and a part of the molecule capable of being metabolized to a reactive group are discussed in terms of the theory of frameshift mutagenesis.
Abstract: 18 Carcinogens, including aflatoxin B1, benzo(a)pyrene, acetylaminofluorene, benzidine, and dimethylamino-trans-stilbene, are shown to be activated by liver homogenates to form potent frameshift mutagens. We believe that these carcinogens have in common a ring system sufficiently planar for a stacking interaction with DNA base pairs and a part of the molecule capable of being metabolized to a reactive group: these structural features are discussed in terms of the theory of frameshift mutagenesis. We propose that these carcinogens, and many others that are mutagens, cause cancer by somatic mutation. A simple, inexpensive, and extremely sensitive test for detection of carcinogens as mutagens is described. It consists of the use of a rat or human liver homogenate for carcinogen activation (thus supplying mammalian metabolism) and a set of Salmonella histidine mutants for mutagen detection. The homogenate, bacteria, and a TPNH-generating system are all incubated together on a petri plate. With the most active compounds, as little as a few nanograms can be detected.
2,146 citations
TL;DR: Tubulin can be purified from guinea pig brain readily and in good yield by two cycles of assembly in glycerol-containing solutions, and is more stable than tubules formed in the absence of these compounds.
Abstract: Microtubule assembly is enhanced by the addition of 1 M sucrose or 4 M glycerol to the reassembly mixture. Tubulin can be purified from guinea pig brain readily and in good yield by two cycles of assembly in glycerol-containing solutions. The tubules assembled in glycerol and sucrose are more stable than tubules formed in the absence of these compounds. Assembly occurs in glycerol or sucrose in the absence of ATP or GTP, but is greatly accelarated by their presence.
1,945 citations
TL;DR: Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules.
Abstract: The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins.
1,641 citations
TL;DR: A test that compares mutagenic killing in deep rough strains with and without DNA excision repair, and a test using forward mutagenesis in a deep rough strain lacking excison repair are described.
Abstract: We previously described a set of four strains of Salmonella typhimurium designed for detecting the various types of mutagens, and showed their utility in detecting a wide variety of carcinogens as mutagens. The lipopolysaccharide that normally coats these bacteria is a barrier to penetration of mutagens to the cell membrane. The set of tester strains has been improved by adding a mutation (rfa: deep rough) that results in a deficient lipopolysaccharide. The techniques for using these strains for detecting mutagens are presented and the tests are shown to be extremely sensitive and convenient. The specificity of frameshift mutagenesis is clarified. As adjuncts to the test with the four strains, we describe a test that compares mutagenic killing in deep rough strains with and without DNA excision repair, and a test using forward mutagenesis in a deep rough strain lacking excision repair.
1,493 citations
TL;DR: The in vitro product directed by globin 9S RNA comigrated precisely with authentic rabbit globin on sodium dodecyl sulfate-polyacrylamide gels during high voltage ionophoresis, and two [(35)S]methionine-labeled tryptic peptides synthesized in vitro comigrated in two dimensions with alphaT5 and betaT5 tryptic amino-acid incorporation from authentic [( 35)S].
Abstract: Extracts prepared from commercially available wheat germ efficiently translate messenger RNAs from either viral or eukaryotic origin. The addition of tobacco mosaic virus RNA stimulated amino-acid incorporation more than 100-times and rabbit globin 9S RNA between 20- and 30-times. The in vitro product directed by globin 9S RNA comigrated precisely with authentic rabbit globin on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, during high voltage ionophoresis two [35S]methionine-labeled tryptic peptides synthesized in vitro comigrated in two dimensions with αT5 and βT5 tryptic peptides from authentic [35S]methionine-labeled rabbit globin.
1,069 citations
TL;DR: Etorphine, the most potent narcotic analgesic known, was labeled with tritium by catalytic exchange and exhibits stereospecific, saturable binding to rat-brain homogenate.
Abstract: Etorphine, the most potent narcotic analgesic known, was labeled with tritium by catalytic exchange. This drug exhibits stereospecific, saturable binding to rat-brain homogenate. At saturation, the stereospecific binding is 0.1-0.15 pmol/mg of protein. Specific binding is inhibited high salt concentrations, sulfhydryl reagents, and proteolytic enzymes, but is unaffected by phospholipases A and C, sodium azide, sodium fluoride, and prostaglandins E1 and E2. Competition for binding of [3H]etorphine correlates with agonist and antagonist potencies. The stable, stereospecific binding of an active narcotic analgesic supports the existence of opiate receptors.
1,051 citations
TL;DR: Preliminary results indicate that the gradient in H. halobium plays the central role in energy coupling attributed to such electrochemical gradients by Mitchell's chemiosmotic theory.
Abstract: The purple membrane of Halobacterium halobium contains only one protein, bacteriorhodopsin, which closely resembles the visual pigments of animals. Light flashes cause a rapid transient shift of its absorption maximum from 560 to 415 nm. This shift is accompanied by release and uptake of protons. Respiring cells acidify the medium in the dark; if they contain purple membrane their O2 consumption is reduced in the light. Starved or anaerobic cells containing purple membrane, in the absence of any apparent source of energy, generate and maintain a proton gradient across the cell membrane as long as they are exposed to light. We postulate that the light-generated proton gradient arises from a vectorial release and uptake of protons by bacteriorhodopsin, which is suitably oriented in the cell membrane and under continuous illumination oscillates rapidly between the long- and short-wavelength form. Preliminary results indicate that the gradient in H. halobium plays the central role in energy coupling attributed to such electrochemical gradients by Mitchell's chemiosmotic theory.
932 citations
TL;DR: Marmoset blood leukocytes transformed in vitro by Epstein-Barr virus regularly release extracellular infectious Epstein- Barr virus with high titers of transforming activity.
Abstract: Marmoset blood leukocytes transformed in vitro by Epstein-Barr virus regularly release extracellular infectious Epstein-Barr virus with high titers of transforming activity. By comparison, human umbilical cord leukocytes and adult human leukocytes transformed by Epstein-Barr virus release either no extracellular infectious virus or small amounts, irregularly.
929 citations
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TL;DR: It is proposed that a cell's life is divided into two fundamentally different parts, some time after mitosis all cells enter a state (A) in which their activity is not directed towards replication, and initiation of cell replication processes is thus random.
Abstract: We propose that a cell's life is divided into two fundamentally different parts. Some time after mitosis all cells enter a state (A) in which their activity is not directed towards replication. A cell may remain in the A-state for any lenght of time, throughout which its probability of leaving A-state remains constant. On leaving A-state, cells enter B-phase in which their activities are deterministic, and directed towards replication. Initiation of cell replication processes is thus random, in the sense that radioactive decay is random. Cell population growth rates are determined by the probability with which cells leave the A-state, the duration of the B-phase, and the rate of cell death. Knowledge of these parameters permits precise calculation of the distribution of intermitotic times within populations, the behavior of synchronized cell cultures, and the shape of labeled mitosis curves.
861 citations
TL;DR: The data show that fibrous caps even of relatively large atheromatous plaques, 0.5 cm or greater in diameter, are composed of cells that produce solely or predominantly one enzyme type, whereas samples of artery wall media and intima as small as 0.1 mm(3) are regularly composed of a mixture of cell types, implying that atherosclerotic plaques in human beings arise by another mechanism.
Abstract: The main cellular elements of atherosclerotic plaques are smooth muscle cells. Because these plaques differ from their precursors in the underlying artery wall in several ways, we have asked the question: Are human atherosclerotic plaques polyclonal or monoclonal in their origin? The X-linked glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in heterozygotic females has been used to obtain an answer. 30 Plaques of different degrees of complexity and 59 samples of normal aorta and iliac artery walls from four females, 25-79 years old, were investigated. The data show that fibrous caps even of relatively large atheromatous plaques, 0.5 cm or greater in diameter, are composed of cells that produce solely or predominantly one enzyme type, whereas samples of artery wall media and intima as small as 0.1 mm3 are regularly composed of a mixture of cell types. If plaques were a response to injury akin to a healing wound, a reaction to a growth stimulant, or formed due to an organization of a mural thrombus, they would be expected to be polyclonal. Hence, the results imply that atherosclerotic plaques in human beings arise by another mechanism. The mechanism compatible with the monoclonal nature of atherosclerotic plaques is mutation, and the likely causes are chemical mutagens or viruses.
TL;DR: Growth of cells in medium containing BrdU for two generations allows fluorometric documentation of the semiconservative distribution of newly replicated DNA between sister chromatids, and regions of sister chromated exchange are demarcated.
Abstract: Fluorescence of the dye 33258 Hoechst bound to chromosomes is partially quenched by the introduction of BrdU into chromosomal DNA. This finding has allowed microfluorometric detection of DNA synthesis in human metaphase chromosomes. Incorporation, shortly before cell harvest, of a pulse of thymidine into chromosomes otherwise substituted with BrdU results in sharply defined foci of bright 33258 Hoechst fluorescence, corresponding to regions of late DNA replication. The latereplicating X chromosome can thereby be clearly identified, and the time course of appearance of its fluorescent bands can be compared with that of its earlier replicating homologue. Growth of cells in medium containing BrdU for two generations allows fluorometric documentation of the semiconservative distribution of newly replicated DNA between sister chromatids, and regions of sister chromated exchange are demarcated. This approach should constitute, in many instances, a convenient, high-resolution fluorometric alternative to autoradiography.
TL;DR: Possible means of testing the hypothesis that the early stages of three-dimensional structure formation (nucleation) occur independently in separate parts of these molecules are discussed.
Abstract: Distinct structural regions have been found in several globular proteins composed of single polypeptide chains. The existence of such regions and the continuity of peptide chain within them, coupled with kinetic arguments, suggests that the early stages of three-dimensional structure formation (nucleation) occur independently in separate parts of these molecules. A nucleus can grow rapidly by adding peptide chain segments that are close to the nucleus in aminoacid sequence. Such a process would generate three-dimensional (native) protein structures that contain separate regions of continuous peptide chain. Possible means of testing this hypothesis are discussed.
TL;DR: The physiological mechanism proposed here for this process posits that at synapses acting according to Hebb's postulate, the receptors for the neurotransmitter are eliminated from the post Synaptic membrane by the transient reversals of the sign of membrane polarization that occur during action potential impulses in the postsynaptic cell.
Abstract: Hebb's postulate of learning envisages that activation or inactivation of extant synaptic contacts in plastic neural networks depends on the synchronous impulse activity of pre- and postsynaptic nerve cells. The physiological mechanism proposed here for this process posits that at synapses acting according to Hebb's postulate, the receptors for the neurotransmitter are eliminated from the postsynaptic membrane by the transient reversals of the sign of membrane polarization that occur during action potential impulses in the postsynaptic cell. But, since the release of neurotransmitter drives the membrane potential of the synaptic zone towards a level about half-way between the negative-inside resting potential and the positive-inside action potential, it would follow that the membrane patches surrounding the receptors of a synapse whose activity has contributed to setting off the postsynaptic impulse would be spared the full extent of the noxious polarity reversal. This mechanism can account for a neurophysiologically documented example of the operation of Hebb's postulate, namely the plasticity of the connections between fourth- and fifth-order neurons in the visual cortex of cats.
TL;DR: Putative cell-surface proteins of tissue-culture cells were identified by lactoperoxidase-catalyzed iodination, a technique that attaches label only to proteins outside the cell membrane, demonstrating that these proteins are cell derived, not contaminating serum proteins.
Abstract: Putative cell-surface proteins of tissue-culture cells were identified by lactoperoxidase-catalyzed iodination, a technique that attaches label only to proteins outside the cell membrane. Evidence is presented that these proteins are cell derived, not contaminating serum proteins. On “normal” cells, which exhibit density dependence of growth, one protein of high molecular weight was particularly readily iodinated. This protein was easily removed by mild proteolytic digestion, and, in virus-transformed cells, was either absent or unavailable for iodination. The possible relevance of these observations to the control of growth in cell culture is discussed.
TL;DR: The smooth muscle-stimulating activity of the endoperoxide ester on the isolated rabbit aortas trip was 4- to 8-times higher than that of the methyl ester of prostaglandin E(2).
Abstract: An earlier proposed endoperoxide intermediate in the biosynthesis of prostaglandins was detected in short-time incubations of arachidonic acid with the microsomal fraction of homogenates of sheep vesicular glands. Conversion of the endoperoxide into prostaglandin E2 was stimulated by reduced glutathione but suppressed by p-mercuribenzoate and N-ethylmaleimide. The methyl ester of an unknown compound was isolated by solvent extraction and thin-layer chromatography after short-time incubation of arachidonic acid with the microsomal fraction and p-mercuribenzoate. This derivative was identical to the methylester of the endoperoxide, as shown by its conversion into the methyl esters of 11-dehydroprostaglandin F2α and prostaglandin E2 by spontaneous rearrangement and its conversion into the methyl ester of prostaglandin F2α by mild chemical reduction. The smooth muscle-stimulating activity of the endoperoxide ester on the isolated rabbit aortas trip was 4- to 8-times higher than that of the methyl ester of prostaglandin E2.
TL;DR: The embryonal carcinoma enzyme resembles the enzymes from kidney and placenta in kinetics of thermal inactivation and sensitivity to the inhibitor L-phenylalanine, but is distinguishable from the alkaline phosphatases of liver and intestine.
Abstract: In tumors and embryoid bodies of mouse teratoma a correlation has been established between specific activity of alkaline phosphatase (EC 3.1.3.1) and content of embryonal carcinoma, the stem cell of the tumor. A histochemical study of embryoid bodies has shown that high levels of the enzyme are confined to embryonal carcinoma. Fifteen tissue culture lines could be classified into three groups: (a) lines identifiable as pluripotential embryonal carcinoma by their morphology, tumorigenicity, and capacity to differentiate in vivo; (b) nullipotential embryonal carcinoma, resembling pluripotential embryonal carcinoma in morphology and malignancy but giving rise to undifferentiated tumors; and (c) lines of apparently nonmalignant somatic cells. Both types of embryonal carcinoma possess levels of alkaline phosphatase 5- to a 100-fold higher than the somatic cell lines. The embryonal carcinoma enzyme resembles the enzymes from kidney and placenta in kinetics of thermal inactivation and sensitivity to the inhibitor L-phenylalanine, but is distinguishable from the alkaline phosphatases of liver and intestine. These findings are discussed in relation to the use of teratoma for the study of cell differentiation.
TL;DR: Comparison of the tracks of wild-type and mutant animals responding to gradients of attractants indicates that sensory receptors in the head alone mediate the orientation response and that the direction of orientation is determined by the lateral motion of the head.
Abstract: The nematode Caenorhabditis elegans is attracted by at least four classes of attractants: by cyclic nucleotides, cAMP and cGMP; by anions, Cl(-), Br(-), I(-); by cations, Na(+), Li(+), K(+), Mg(+); and by alkaline pH values. The nematode's behavioral response to gradients of these attractants involves orientation and movement up the gradient, accumulation, and then habitutation. Comparison of the tracks of wild-type and mutant animals responding to gradients of attractants indicates that sensory receptors in the head alone mediate the orientation response and that the direction of orientation is determined by the lateral motion of the head. Therefore, the orientation response is a klinotaxis.
TL;DR: Various species of Streptomyces possess aminoglycoside-modifying enzymes that catalyze reactions identical to those catalyzed by enzymes found in gram-negative bacteria containing R(antibiotic resistance)-factors, suggesting the possibility of an evolutionary relationship between these enzymes and the R-factors.
Abstract: Various species of Streptomyces possess aminoglycoside-modifying enzymes. Streptomyces kanamyceticus contains an enzyme that acetylates the 6′-amino group of kanamycin A and B, gentamicin C1a, and neomycin. Streptomyces spectabilis produces an enzyme that acetylates the 2′-amino group of the hexose ring of gentamicin C1a. These enzymes catalyze reactions identical to those catalyzed by enzymes found in gram-negative bacteria containing R(antibiotic resistance)-factors. The discovery of these enzymes suggests the possibility of an evolutionary relationship between the aminoglycosideinactivating enzymes (produced by resistance determinants) in bacteria containing R-factors and similar enzymes found in the actinomycetes.
TL;DR: This model represents an extension of the Lotka-Volterra model of competition; it adds a fourth parameter that controls the degree of nonlinearity in intraspecific growth regulation and represents a similar Extension of the logistic model of population growth.
Abstract: Very precise data on the dynamics of a competitive system of two species of Drosophila have been obtained. By a curvilinear regression approach, analytical models of competition have been fitted. By statistical and biological criteria of simplicity, reality, generality, and accuracy, the best of these models has been chosen. This model represents an extension of the Lotka-Volterra model of competition; it adds a fourth parameter that controls the degree of nonlinearity in intraspecific growth regulation. It represents a similar extension of the logistic model of population growth.
TL;DR: Results suggest that the antiviral activity of Virazole might be due to the inhibition of GMP biosynthesis in the infected cell at the step involving the conversion of IMP to xanthosine 5'-phosphate, which would result in inhibition of the synthesis of vital viral nucleic acid.
Abstract: The antiviral activity of the synthetic nucleoside, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), against measles virus in Vero cell cultures was substantially reversed by xanthosine, guanosine, and to a slightly lesser extent by inosine. Virazole 5'-phosphate was subsequently found to be a potent competitive inhibitor of inosine 5'-phosphate dehydrogenase (IMP:NAD(+) oxidoreductase, EC 1.2.1.14) isolated from Escherichia coli (K(m) = 1.8 x 10(-5) M) with a K(i) of 2.7 x 10(-7) M. Guanosine 5'-phosphate (GMP) was a competitive inhibitor of this enzyme with a K(i) of 7.7 x 10(-5) M. Virazole 5'-phosphate was similarly active against IMP dehydrogenase isolated from Ehrlich ascites tumor cells, with a K(i) of 2.5 x 10(-7) M. The K(m) for this enzyme was 1.8 x 10(-5) M, and the K(i) for GMP was 2.2 x 10(-4) M. These results suggest that the antiviral activity of Virazole might be due to the inhibition of GMP biosynthesis in the infected cell at the step involving the conversion of IMP to xanthosine 5'-phosphate. This inhibition would consequently result in inhibition of the synthesis of vital viral nucleic acid.
TL;DR: The nucleotides ppGpp and pppGpp accumulate when wild-type, but not rel(-), strains of Escherichia coli are starved for required amino acids.
Abstract: The nucleotides ppGpp and pppGpp accumulate when wild-type, but not rel-, strains of Escherichia coli are starved for required amino acids. These compounds are synthesized on ribosomes, in the presence of the product of the rel gene, from GDP and GTP; ATP is used as the phosphate donor. The signal for making these compounds is the presence of an uncharged tRNA in the ribosomal acceptor site. These compounds are not accumulated if the ribosomes are actively engaged in protein synthesis.
TL;DR: It is suggested that all cells possess multiple structural genes capable of coding for transforming factors which can release the cell from its normal constraints on growth.
Abstract: A general hypothesis of carcinogenesis is proposed consisting of the following features: (1) It is suggested that all cells possess multiple structural genes (Tr) capable of coding for transforming factors which can release the cell from its normal constraints on growth. (2) In adult cells they are suppressed by diploid pairs of regulatory genes and some of the transforming genes are tissue specific. (3) The Tr loci are temporarily activated at some stage of embryogenesis and possibly during some stage of the cell cycle in adult cells. (4) Spontaneous tumors, or tumors induced by chemicals or radiation, arise as the result of a double mutation of any set of regulatory genes releasing the suppression of the corresponding Tr genes and leading to transformation of the cell. (5) Autosomal dominant hereditary tumors, such as retinoblastoma, are the result of germ-line inheritance of one inactive regulatory gene. Subsequent somatic mutation of the other regulatory gene leads to tumor formation. (6) The Philadelphia chromosome produces inactivation of one regulatory gene by position effect. A somatic mutation of the other leads to chronic myelogenous leukemia. (7) Oncogenic viruses evolved by the extraction of host Tr genes with their conversion to viral transforming genes. As a result, in addition to the above mechanisms, tumors may also be produced by the reintroduction of these genes into susceptible host cells.
TL;DR: Cytochrome oxidase (EC 1.3.1) isolated from beef-heart mitochondria with an appropriate phospholipid content forms vesicular structures, interpreted as evidence for a boundary of immobilized lipid between the hydrophobic protein and adjacent fluid bilayer regions in this membrane model system.
Abstract: Cytochrome oxidase (EC 1.9.3.1) isolated from beef-heart mitochondria with an appropriate phospholipid content forms vesicular structures. Lipid-protein interactions in this model membrane system were studied with the lipid spin label, 16-doxylstearic acid. As the phospholipid/protein ratio is varied, two spectral components are observed. At low phospholipid/protein ratios (≤0.19 mg of phospholipid per mg of protein) the lipid spin label is highly immobilized. At higher phospholipid content an additional component characteristic of fluid lipid bilayers is evident. By summation of digitalized spectra and subsequent integration it was shown that all composite spectra could be approximated by assuming only two components are present, and that the amount of phospholipid bound to the protein is independent of the extent of the fluid bilayer region. The experimentally determined amount of phospholipid for maximum occupancy of protein-bound sites is about 0.2 mg of phospholipid per 1.0 mg of protein. Calculations show that this ratio is consistent with a single layer of phospholipid surrounding the protein complex. The data are interpreted as evidence for a boundary of immobilized lipid between the hydrophobic protein and adjacent fluid bilayer regions in this membrane model system.
TL;DR: The sequence of RNA transcription copies of this fragment is determined and the sequence has 2-fold symmetry regions; the two longest are separated by one turn of the DNA double helix.
Abstract: The lac repressor protects the lac operator against digestion with deoxyribonuclease. The protected fragment is double-stranded and about 27 base-pairs long. We determined the sequence of RNA transcription copies of this fragment and present a sequence for 24 base pairs. It is: 5′--T G G A A T T G T G A G C G G A T A A C A A T T 3′ 3′--A C C T T A A C A C T C G C C T A T T G T T A A 5′ The sequence has 2-fold symmetry regions; the two longest are separated by one turn of the DNA double helix.
TL;DR: Potencies of opiates and their antagonists in displacing [(3)H]naloxone binding parallel their pharmacological potencies.
Abstract: [3H]Naloxone, a potent opiate antagonist, binds stereospecifically to opiate-receptor sites in rat-brain tissue. The binding is time, temperature, and pH dependent and saturable with respect to [3H]naloxone and tissue concentration. The [3H]naloxone-receptor complex formation is bimolecular with a dissociation constant of 20 nM. 15 Opiate agonists and antagonists compete for the same receptors, whose density is 30 pmol/g. Potencies of opiates and their antagonists in displacing [3H]naloxone binding parallel their pharmacological potencies.
TL;DR: The demonstration of apparently identical kinetic properties of the reductase activity of control and mutant cells, coupled with the evidence that this enzyme is normally regulated not by allosteric effectors but by alterations in enzyme synthesis and degradation, suggests that the primary genetic abnormality does not involve the structural gene for the enzyme itself, but a hitherto unidentified gene whose product is necessary for mediation of feedback control by lipoproteins.
Abstract: The homozygous form of the autosomal dominant disorder, familial hypercholesterolemia, is characterized by the presence in children of profound hypercholesterolemia, cutaneous planar xanthomas, and rapidly progressive coronary vascular disease that usually results in death before age 30 years. Cultured skin fibroblasts from three unrelated subjects with this disorder showed 40- to 60-fold higher activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-controlling enzyme in cholesterol biosynthesis, when compared with fibroblasts of seven control subjects. Enhanced enzyme activity resulted from a complete absence of normal feedback suppression by low-density lipoproteins, which led to a marked overproduction of cholesterol by the mutant cells. The demonstration of apparently identical kinetic properties of the reductase activity of control and mutant cells, coupled with the evidence that this enzyme is normally regulated not by allosteric effectors but by alterations in enzyme synthesis and degradation, suggests that the primary genetic abnormality does not involve the structural gene for the enzyme itself, but a hitherto unidentified gene whose product is necessary for mediation of feedback control by lipoproteins. The fibroblasts of two obligate heterozygotes, the parents of one of the homozygotes, showed a pattern of enzyme regulation intermediate between that of controls and homozygotes.
TL;DR: The proposed model has several implications for studies of the initial events of mitogenesis in lymphocytes as well as for cell-cell interactions in general.
Abstract: An analysis of the inhibition by concanavalin A of the mobility of lymphocyte surface receptors is used to construct an hypothesis on membrane receptor-cytoplasmic interactions. It is proposed that binding of multivalent lectins alters the interaction of an assembly of colchicine-binding proteins with lectin receptors and other receptors, and reciprocally that the state of the colchicine-binding assembly alters the mobility and distribution of surface receptors on the cell membrane. Observations of the effect of colchicine and related drugs on the inhibition of receptor mobility by concanavalin A lend support to this hypothesis. The proposed model has several implications for studies of the initial events of mitogenesis in lymphocytes as well as for cell-cell interactions in general.
TL;DR: Con A shows a greater capacity than succinyl-Con A to agglutinate sheep erythrocytes and to inhibit cap formation by immunoglobulin receptors on spleen cells, and at low concentrations, Con A induced its glycoprotein receptors to form caps, but succ Vinyl A did not induce cap formation.
Abstract: Chemical derivatization of tetrameric concanavalin A (Con A) with succinic anhydride or acetic anhydride converts the protein to a dimeric molecule without altering its carbohydrate-binding specificity. At low concentrations, the dose-response curves for the mitogenic stimulation of mouse spleen cells by native Con A and succinyl-Con A are similar. Above lectin concentrations of 10 μg/ml, however, the response to Con A is diminished, while that for succinyl-Con A does not decrease until much higher doses are reached. We have attributed this difference mainly to the higher rate of cell death induced by the native Con A molecule. Con A also shows a greater capacity than succinyl-Con A to agglutinate sheep erythrocytes and to inhibit cap formation by immunoglobulin receptors on spleen cells. Moreover, at low concentrations, Con A induced its glycoprotein receptors to form caps, but succinyl-Con A did not induce cap formation. Addition of antibodies directed against Con A to succinyl-Con A bound on cells restored the properties of agglutination, inhibition of immunoglobulin receptor cap formation, and induction of cap formation by Con A receptors. Similar results have been obtained for acetyl-Con A. These data suggest that the altered biological activities of succinyl-Con A and acetyl-Con A are attributable to their reduced valence.
TL;DR: The regional localization of strychnine binding in the central nervous system correlates closely with endogenous glycine concentrations, and in subcellular fractionation experiments, stryhnine binding is most enhanced in synaptic-membrane fractions.
Abstract: [3H]Strychnine binds to synaptic-membrane fractions of the spinal cord in a selective fashion, indicating an interaction with postsynaptic glycine receptors. Displacement of strychnine by glycine and other amino acids parallels their glycine-like neurophysiologic activity. The regional localization of strychnine binding in the central nervous system correlates closely with endogenous glycine concentrations. In subcellular fractionation experiments, strychnine binding is most enhanced in synaptic-membrane fractions. Strychnine binding is saturable, with affinity constants for glycine and strychnine of 10 and 0.03 μM, respectively.