Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 1979"

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

Abstract: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

Mathematical model for studying genetic variation in terms of restriction endonucleases

Abstract: A mathematical model for the evolutionary change of restriction sites in mitochondrial DNA is developed. Formulas based on this model are presented for estimating the number of nucleotide substitutions between two populations or species. To express the degree of polymorphism in a population at the nucleotide level, a measure called "nucleotide diversity" is proposed.

Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.

Abstract: pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.

Rapid evolution of animal mitochondrial DNA.

Abstract: Mitochondrial DNA was purified from four species of higher primates (Guinea baboon, rhesus macaque, guenon, and human) and digested with 11 restriction endonucleases. A cleavage map was constructed for the mitochondrial DNA of each species. Comparison of the maps, aligned with respect to the origin and direction of DNA replication, revealed that the species differ from one another at most of the cleavage sites. The degree of divergence in nucleotide sequence at these sites was calculated from the fraction of cleavage sites shared by each pair of species. By plotting the degree of divergence in mitochondrial DNA against time of divergence, the rate of base substitution could be calculated from the initial slope of the curve. The value obtained, 0.02 substitutions per base pair per million years, was compared with the value for single-copy nuclear DNA. The rate of evolution of the mitochondrial genome appears to exceed that of the single-copy fraction of the nuclear genome by a factor of about 10. This high rate may be due, in part, to an elevated rate of mutation in mitochondrial DNA. Because of the high rate of evolution, mitochondrial DNA is likely to be an extremely useful molecule to employ for high-resolution analysis of the evolutionary process.

Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

Abstract: We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.

Binding Site on Macrophages that Mediates Uptake and Degradation of Acetylated Low Density Lipoprotein, Producing Massive Cholesterol Deposition

Abstract: Resident mouse peritoneal macrophages were shown to take up and degrade acetylated 125I-labeled low density lipoprotein (125I-acetyl-LDL) in vitro at rates that were 20-fold greater than those for the uptake and degradation of 125I-LDL. The uptake of 125I-acetyl-LDL and its subsequent degradation in lysosomes were attributable to a high-affinity, trypsin-sensitive, surface binding site that recognized acetyl-LDL but not native LDL. When 125I-acetyl-LDL was bound to this site at 4°C and the macrophages were subsequently warmed to 37°C, 75% of the cell-bound radioactivity was degraded to mono[125I]iodotyrosine within 1 hr. The macrophage binding site also recognized maleylated LDL, maleylated albumin, and two sulfated polysaccharides (fucoidin and dextran sulfate) indicating that negative charges were important in the binding reaction. A similar binding site was present on rat peritoneal macrophages, guinea pig Kupffer cells, and cultured human monocytes but not on human lymphocytes or fibroblasts, mouse L cells or Y-1 adrenal cells, or Chinese hamster ovary cells. Uptake and degradation of acetyl-LDL via this binding site stimulated cholesterol esterification 100-fold and produced a 38-fold increase in the cellular content of cholesterol in mouse peritoneal macrophages. Although the physiologic significance, if any, of this macrophage uptake mechanism is not yet known, we hypothesize that it may mediate the degradation of denatured LDL in the body and thus serve as a “backup” mechanism for the previously described receptor-mediated degradation of native LDL that occurs in parenchymal cells. Such a scavenger pathway might account for the widespread deposition of LDL-derived cholesteryl esters in macrophages of patients with familial hypercholesterolemia in whom the parenchymal cell pathway for LDL degradation is blocked, owing to a genetic deficiency of receptors for native LDL.

Growth of a rat neuroblastoma cell line in serum-free supplemented medium.

Abstract: The rat neuroblastoma B104 cell line, which originated in the central nervous system, was able to proliferate in the absence of serum in synthetic medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine. When added individually, each supplement had little or no effect; however, in combination there was a marked synergistic effect on cell number. The cells attained the same saturation density in this medium as in medium with 10% fetal calf serum added. More extensive process formation was observed in the supplemented medium, and other differentiated properties were retained as well.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.

Abstract: Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the concentration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immunologically tolerated "tissue" for wound healing and as a model for studying fibroblast function are discussed.

Universal variational functionals of electron densities, first-order density matrices, and natural spin-orbitals and solution of the v-representability problem

Abstract: Universal variational functionals of densities, first-order density matrices, and natural spin-orbitals are explicitly displayed for variational calculations of ground states of interacting electrons in atoms, molecules, and solids. In all cases, the functionals search for constrained minima. In particular, following Percus [Formula: see text] is identified as the universal functional of Hohenberg and Kohn for the sum of the kinetic and electron—electron repulsion energies of an N-representable trial electron density ρ. Q[ρ] searches all antisymmetric wavefunctions Ψρ which yield the fixed. ρ. Q[ρ] then delivers that expectation value which is a minimum. Similarly, [Formula: see text] is shown to be the universal functional for the electron—electron repulsion energy of an N-representable trial first-order density matrix γ, where the actual external potential may be nonlocal as well as local. These universal functions do not require that a trial function for a variational calculation be associated with a ground state of some external potential. Thus, the v-representability problem, which is especially severe for trial first-order density matrices, has been solved. Universal variational functionals in Hartree—Fock and other restricted wavefunction theories are also presented. Finally, natural spin-orbital functional theory is compared with traditional orbital formulations in density functional theory.

Preparative and analytical purification of DNA from agarose.

Abstract: Two procedures were developed for removing DNA from agarose after electrophoretic separation of DNA fragments according to size. Both involve dissolving the DNA-containing agarose in NaI. The preparative technique uses binding of DNA to glass in the presence of NaI. The method is rapid and convenient, and DNA of all molecular weight ranges can be recovered in high yield and without degradation. The DNA is free of agarose and remains susceptible to digestion by restriction enzymes. The analytical technique uses selective precipitation of DNA with acetone and has been adapted to molecular hybridization scans of sequences in agarose gels. The sequence-monitoring system is quantitative, directly measuring the proportion of the probe complementary to a given DNA fragment and vice versa. It is especially suitable for analyzing restriction enzyme digests of DNA in mapping experiments.

High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules

Abstract: A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.

Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene.

Abstract: L-[U-14C]Methionine fed to apple tissue was efficiently converted to ethylene when the tissue was incubated in air. In nitrogen, however, it was not metabolized to ethylene but was instead converted to 1-aminocyclopropane-1-carboxylic acid (ACC). When apple tissues were fed with L-[methyl-14C]methionine or L-[35S]methionine and incubated in nitrogen, radioactivity was found subsequently in methylthioribose. This suggests that methionine is first converted to S-adenosylmethionine which is in turn fragmented to ACC and methylthioadenosine. Methylthioadenosine is then hydrolyzed to methylthioribose. The conclusion that ACC is an intermediate in the conversion of methionine to ethylene is based on the following observations: Labeled ACC was efficiently converted to ethylene by apple tissue incubated in air; the conversion of labeled methionine to ethylene was greatly decreased in the presence of unlabeled ACC, but the conversion of labeled ACC to ethylene was little affected by the presence of unlabeled methionine; and 2-amino-4-(2′-aminoethoxy)trans-3-butenoic acid, a potent inhibitor of pyridoxal phosphate-mediated enzyme reactions, greatly inhibited the conversion of methionine to ethylene but did not inhibit conversion of ACC to ethylene. These data indicate the following sequence for the pathway of ethylene biosynthesis in apple tissue: methionine → S-adenosylmethionine → ACC → ethylene. A possible mechanism accounting for these reactions is presented.

Growth of cultured human epidermal cells into multiple epithelia suitable for grafting.

Abstract: Owing to several recent developments, the cultivability of epidermal keratinocytes, particularly those of the human, has been greatly improved. Under the conditions used, single cultured cells generate stratified colonies that ultimately fuse and form an epithelium that is reasonable approximation of the epidermis. It will be shown here that large amounts of cultured epithelium can be generated from a small piece of epidermis in a short time. We wish to bring to the attention of surgeons and cell biologists the possibility of using culture-grown epithelium derived from the same individual to restore defects in the epidermis.

DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells

Abstract: In this report, we demonstrate the feasibility of transforming mouse cells deficient in adenine phosphoribosyltransferase (aprt; AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) to the aprt+ phenotype by means of DNA-mediated gene transfer. Transformation was effected by using unfractionated high molecular weight genomic DNA from Chinese hamster, human, and mouse cells and restriction endonuclease-digested DNA from rabbit liver. The transformation frequency observed was between 1 and 10 colonies per 10(6) cells per 20 microgram of donor DNA. Transformants displayed enzymatic activity that was donor derived as demonstrated by isoelectric focusing of cytoplasmic extracts. These transformants fall into two classes: those that are phenotypically stable when grown in the absence of selective pressure and those that are phenotypically unstable under the same conditions.

Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus

Abstract: Patients with systemic lupus erythematosus often possess antibodies against two nuclear antigens called Sm and RNP (ribonucleoprotein). We have established the molecular identity of these antigens by analyzing immune precipitates of nuclear extracts from mouse Ehrlich ascites cells labeled with 32P and 35S. Anti-Sm serum selectively precipitates six small nuclear RNA molecules (snRNAs); anti-RNP serum reacts with only two of these; and a third serum, characterized as mostly anti-RNP, precipitates a subset of three snRNA bands. Three of the six RNAs are identified by fingerprint analysis as the previously characterized and highly abundant nucleoplasmic snRNA species U1a (171 nucleotides), U1b, and U2 (196 nucleotides). The other three RNAs (U4, U5, and U6) likewise are uridine rich and contain modified nucleotides, but they are smaller, with lengths of about 145, 120, and 95 residues, respectively. Each of the six snRNAs is complexed with and apparently antigenic by virtue of association with specific proteins. All three sera precipitate an identical complement of seven different polypeptides ranging in molecular weight from 12,000 to 35,000; these proteins are abundant in nuclear extracts, but are neither histones nor the major polypeptides comprising the 30S heterogeneous nuclear RNP particles of mammalian nuclei. Our data argue that each of the six snRNAs exists in a separate small nuclear ribonucleoprotein (snRNP) complex with a total molecular weight of about 175,000. We find that human antisera also precipitate snRNAs from a wide range of vertebrate species and from arthropods. We discuss the antigenic snRNPs in relation to the published literature on snRNAs and nuclear RNPs and consider possible functions of snRNPs in nuclear processes.

Separation of functional subsets of human T cells by a monoclonal antibody.

Abstract: A monoclonal antibody was produced to human peripheral blood T cells. This hybridoma antibody, termed OKT4, was reactive by indirect immunofluorescence with only 55-60% of the peripheral blood T cell population (OKT4+) and unreactive with normal B cells, null cells, and macrophages. The OKT4- T cell population contained the previously described TH2+ subset that has been shown to contain cytotoxic/suppressor cells. With cell-sorter separation of OKT4+ and OKT4- cells, it was shown that these T cell subsets were functionally discrete. Both gave proliferative responses with concanavalin A, alloantigens, and phytohemagglutinin although OKT4+ cells were much more responsive to the latter. OKT4+ cells alone responded to soluble antigens whereas OKT4- cells alone were cytotoxic after alloantigenic sensitization of unfractionated T cells. However, both OKT4+ and OKT4- cells were required during sensitization for optimal development of cytotoxicity. These data suggest that the OKT4+ subset represents a helper population and that the OKT4- subset contains the cytotoxic effector population. OKT4 could be a valuable reagent for determining alterations of these functional subsets in human diseases.

Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus

Abstract: The experimental work described tested the prosposition that the suprachiasmatic nucleus of the hypothalamus is an autonomous circadian pacemaker. Simultaneous recording from two extracellular electrodes indicated neural (multiple unit) activity at two sites in the brain, one of which is in or near the suprachiasmatic nucleus and the other in one of many other brain locations. Both sites in intact rats displayed clear circadian rhythmicity of spontaneous neural activity. In experimental animals, a Halasz knife was used to create an island of hypothalamic tissue that contained the suprachiasmatic nuclei. In such animals that were also blinded by bilateral ocular enucleation, circadian rhythmicity was lost at all brain locations recorded outside the island, but it persisted within the island that contained the suprachiasmatic nuclei. The rhythmicity of the island is thus not dependent on afferent inputs from elsewhere in the brain.

Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester.

Abstract: When suspension cultures of human promyelocytic leukemia cells (line HL60) were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA; 1.6-160 nM), more than 80% of the cells adhered to the plastic substrate within 24 hr. Within the same time period the immature azurophilic granulations typical of HL60 promyelocytic cells disappeared and the nuclear chromatin became more condensed, but the nucleolus was retained. The attached cells stopped dividing and synthesizing DNA. The phenomenon was irreversible and independent of the continuous presence of TPA. Approximately 60% of the untreated cells and of TPA-treated cells bore surface Fc receptors for IgG. Under the experimental conditions used, about 10% of the TPA-treated cells were also able to phagocytize IgG-coated erythrocytes and more than 80% were able to phagocytize latex beads, but untreated controls were unable to do so. Cellular levels of NADase, acid phosphatase, and non-specific esterase were markedly increased after treatment with TPA, whereas little or no increase was seen after treatment with dimethyl sulfoxide (Me2SO), a drug that induces myeloid differentiation of HL60 cells. Peroxidase activity was lower in TPA-treated and Me2SO-treated cells than in HL60 cells. More lysozyme was found in the medium of TPA-treated cells than in the medium of untreated or Me2SO-treated cells. These data indicate that, after treatment with TPA, human promyelocytic leukemia cells can differentiate into cells that have several characteristics of macrophages.

Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences

Abstract: The lactose structural genes, without the lactose promoter, have been incorporated into the bacteriophage Mu genome to form a Mu-lac specialized transducing phage. This phage also carries a gene encoding resistance to ampicillin (Ap)[Mu(Ap, lac)]. After infection and upon establishment of lysogeny, the Mu(Ap, lac) genome can integrate into apparently random sites in the Escherichia coli chromosome. When integration occurs within a gene in the orientation of its transcription, the lactose structural genes are so situated that they become expressed solely from the promoter of that gene. Thus, expression of the lactose genes of Mu(Ap, lac) can be used as an assay for transcription of that gene and for functional and mutational studies of gene regulation.

Monoclonal antibody to a plasma membrane antigen of neurons

Abstract: Fusion of spleen cells from a mouse immunized with chicken embryo retina cells with clonal mouse myeloma cells yielded a lymphocyte hybrid cell line that produced antibody that bound to neural tissue such as retina, brain, spinal cord, and dorsal root ganglia but not to other tissues tested. The antigen was shown by indirect immunofluorescence to be associated with plasma membranes of most, or all, neuron cell bodies in chicken retina, but little or no antigen was detected on axons or dendrites, Muller cells, or retina pigment cells. The activity of antigen A2B5 is relatively stable at 100 degrees C, is insensitive to trypsin, exhibits the solubility properties of a ganglioside, and is destroyed by neuraminidase. Antibody A2B5 cytotoxicity against retina cells is inhibited by a GQ ganglioside fraction from bovine brain (estimated half-maximal inhibition at 0.2 microM) or by N-acetylneuraminic acid (half-maximal inhibition at 5000 microM) but not by other purified gangliosides tested. These results suggest that the antigen is a complex ganglioside in plasma membranes of retina neuron cell bodies but not axons or dendrites.

Galactosamine-induced sensitization to the lethal effects of endotoxin.

Abstract: Treatment of rabbits, rats, and mice with D-galactosamine increased their sensitivity to the lethal effects of lipopolysaccharide several thousand fold. The susceptibility of the animals was highest when the lipopolysaccharide was injected together with galactosamine and decreased successively when injection was carried out 1, 2, and 3 hr later. Sensitization was absent when the lipopolysaccharide was administered 1 hr before or 4 hr after galactosamine. The onset of lethality after treatment with galactosamine and lipopolysaccharide occurred faster than with lipopolysaccharide alone; usually all animals died 5-9 hr later. The galactosamine-induced sensitization to lipopolysaccharide could be reversed by uridine which is known to inhibit the early biochemical alterations induced by the amino sugar in the hepatocytes. Although galactosamine is known to exhibit hepatotoxic activity inducing ultimate necrosis of the hepatocytes, the data so far suggests that the sensitization to lipopolysaccharide is related only to the early metabolic effects of the hexosamine.

Dual control of cell growth by somatomedins and platelet-derived growth factor

Abstract: Quiescent BALB/c 3T3 cells exposed briefly to a platelet-derived growth factor (PDGF) become "competent" to replicate their DNA but do not "progress" into S phase unless incubated with growth factors contained in platelet-poor plasma. Plasma from hypophysectomized rats is deficient in progression activity; it does not stimulate PDGF-treated competent cells to synthesize DNA, demonstrating that somatomedin C is required for progression. Various growth factors were tested for progression activity and competence activity by using BALB/c 3T3 tissue culture assays. Multiplication stimulating activity and other members of the somatomedin family of growth factors are (like somatomedin C) potent mediators of progression. Other mitogenic agents, such as fibroblast growth factor, are (like PDGF) potent inducers of competence. Growth factors with potent progression activity have little or no competence activity and vice versa. In contrast, simian virus 40 provides both competence and progression activity. Coordinate control of BALB/c 3T3 cell growth in vitro by competence factors and somatomedins may be a specific example of a common pattern of growth regulation in animal tissues.

Leukotriene C: a slow-reacting substance from murine mastocytoma cells.

Abstract: Murine mastocytoma cells treated with calcium ionophore A23187 produced a slow-reacting substance (SRS) that caused guinea pig ileum to contract. The response was reversed by the SRS antagonist FPL 55712. On the basis of isotope incorporation experiments, spectroscopy, and chemical degradations, the SRS was identified as a cysteine-containing derivative of 5-hydroxy-7,9,11,14-icosatetraenoic acid. This amino acid was attached in thioether linkage at C-6. The SRS is structurally related to previously identified epoxy and dihydroxy metabolites of arachidonic acid in leukocytes. A common feature is the presence of a conjugated triene, and the name "leukotriene" has been introduced to designate these compounds. Leukotriene A (5,6-epoxy-7,9,11,14-icosatetraenoic acid) is an intermediate in the formation of leukotriene B (5,12-dihydroxy-6,8,10,14-icosatetraenoic acid) and is proposed to be a precursor also of leukotriene C, which is the SRS identified here.

Dynorphin-(1-13), an extraordinarily potent opioid peptide

Abstract: We describe the opioid properties of a tridecapeptide, the sequence of which corresponds to the NH2-terminal sequence of dynorphin, a novel porcine pituitary endorphin. It contains [Leu]enkephalin. In the guinea pig ileum longitudinal muscle preparation it is about 700 times more potent than [Leu]enkephalin. Its effects in this tissue are blocked completely by naloxone, but the apparent affinity of naloxone is 1/13th that for blockade of [Leu]enkephalin or normorphine. In the mouse vas deferens, this peptide is 3 times more potent than [Leu]enkephalin. Well-washed rat brain membranes degrade the peptide rapidly, suggesting the presence of a membrane-bound degradative enzyme. The peptide displays considerable immunoreactivity in assays with antisera that have been used for the immunohistochemical localization of [Leu]enkephalin. The remarkable enhancement of the potency of [Leu]enkephalin by the COOH-terminal extension -Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-OH suggests new interpretations concerning the structure of opiate receptors and the function of the enkephalin pentapeptides.

Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties

Abstract: Platelets enzymatically convert prostaglandin H3 (PGH3) into thromboxane A3. Both PGH2 and thromboxane A2 aggregate human platelet-rich plasma. In contrast, PGH3 and thromboxane A3 do not. PGH3 and thromboxane A3 increase platelet cyclic AMP in platelet-rich plasma and thereby: (i) inhibit aggregation by other agonists, (ii) block the ADP-induced release reaction, and (iii) suppress platelet phospholipase-A2 activity or events leading to its activation. PGI3 (Δ17-prostacyclin; synthesized from PGH3 by blood vessel enzyme) and PGI2 (prostacyclin) exert similar effects. Both compounds are potent coronary relaxants that also inhibit aggregation in human platelet-rich plasma and increase platelet adenylate cyclase activity. Radioactive eicosapentaenoate and arachidonate are readily and comparably acylated into platelet phospholipids. In addition, stimulation of prelabeled platelets with thrombin releases comparable amounts of eicosapentaenoate and arachidonate, respectively. Although eicosapentaenoic acid is a relatively poor substrate for platelet cyclooxygenase, it appears to have a high binding affinity and thereby inhibits arachidonic acid conversion by platelet cyclooxygenase and lipoxygenase. It is therefore possible that the triene prostaglandins are potential antithrombotic agents because their precursor fatty acids, as well as their transformation products, PGH3, thromboxane A3, and PGI3, are capable of interfering with aggregation of platelets in platelet-rich plasma.

Long-term culture of capillary endothelial cells

Abstract: Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells in primary culture. Cultured bovine capillary endothelial cells produce Factor VIII antigen and angiotensin-converting enzyme, but do not have Weibel-Palade bodies. Human cells do contain Weibel-Palade bodies. Capillary endothelial cells are distinguished from aortic endothelial cells by their requirement for conditioned medium. Bovine capillary endothelial cells in regular medium grow slowly with a mean doubling time of 67 hr and eventually die. In tumor-conditioned medium, these cells grow rapidly with a doubling time of 28 hr and continue to proliferate for as long as the tumor-conditioned medium is present. In contrast, bovine aortic endothelial cells grow as rapidly in regular medium as in tumor-conditioned medium. This method allows the production of pure capillary endothelial cells that may prove useful for studies of tumor angiogenesis, metastatic mechanisms, and the role of capillary endothelium in other pathologic states.

Expression in Escherichia coli of chemically synthesized genes for human insulin

Abstract: Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli beta-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from beta-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.

Simultaneous hermaphroditism and sexual selection.

Abstract: Theory about the evolution of sexual behavior in dioecious species is based on the general assumption that egg production is limited by a female's ability to garner resources to make eggs, not by a lack of sperm to fertilize them. Reproductive success for males is thus limited by access to females (and their eggs). I suggest that egg production by simultaneous hermaphrodites also obeys this principle-that fertilized egg production by an individual is not limited by sperm availability, but by resources allocated to eggs. If true, this suggests that sperm competition (reproduction success through male function) and a form of male-female conflict have played important roles in the evolution of hermaphroditism.

Fluorescent phallotoxin, a tool for the visualization of cellular actin

Abstract: A fluorescent derivative of phalloidin has been synthesized possessing high affinity to filamentous actin. This compound was used for visualization of actin-containing structures in eukaryotic nonmuscle cells. Due to its low molecular weight (1250), fixation for formaldehyde was sufficient to render the membrane permeable for the labeled peptide. Bundles of microfilaments are the predominant pattern in the flat rat kangaroo PtK1 cells, whereas a net of concentric fibers characterizes the more spherical bovine kidney MDBK cells. Specificity of staining was confirmed by competition experiments with unlabeled phalloidin.

Arachidonic Acid Metabolism in Polymorphonuclear Leukocytes: Effects of Ionophore A23187

Abstract: Addition of arachidonic acid and the divalent cation ionophore A23187 to a suspension of human peripheral blood polymorphonuclear leukocytes led to the formation of (5S)-hydroxy-6,8,11,14-icosatetraenoic acid, (15S)-hydroxy-5,8,11,13-icosatetraenoic acid, and (5S,12R)-dihydroxy-6,8,10,14-icosatetraenoic acid. A method based on high-pressure liquid chromatography has been developed for assay of these metabolites. The addition of arachidonic acid to human polymorphonuclear leukocytes always resulted in formation of the isomeric monohydroxy acids. However, cells prepared from blood of different subjects were found to vary with respect to formation of the 5,12-dihydroxy acid. Addition of the ionophore alone strongly stimulated the formation of the 5-monohydroxy acid and more specifically the 5,12-dihydroxy acid from endogenous arachidonic acid. In all experiments performed the formation of the 5-hydroxy acid and the 5,12-dihydroxy acid was maximally stimulated when both arachidonic acid and the ionophore were added to the incubation mixture. Under these conditions, stimulation of 40-fold or more of the formation of both compounds was observed. The data demonstrate that, in addition to causing release of endogenous substrate, the ionophore also activated the enzymatic system involved in the further transformations of arachidonic acid. This finding raises the possibility that this pathway of arachidonic acid metabolism is involved in the biological response (e.g., release of lysosomal enzymes, the slow reacting substance of anaphylaxis, and chemotactic factors) of leukocytes to A23187 and other stimuli.