Quaternary enhancement in binding of oxygen by human hemoglobin. From an experimental study on the linkage between dimer-tetramer association and oxygen binding in human hemoglobin it is found that triliganded tetramers alpha(2)beta(2)(O(2))(3) have an affinity for oxygen that is significantly higher than that of alpha(1)beta(1) dimers (superscripts denote intersubunit contacts). This conclusion is based upon a newly determined series of accurate oxygen binding isotherms, which were analyzed in conjunction with independently determined values of the dimer-tetramer equilibrium constants in the unliganded and fully oxygenated states [Ip, S. H. C. & Ackers, G. K. (1977) J. Biol. Chem. 252, 82-87]. The results imply that in the molecule alpha(2)beta(2)(O(2))(3) the interactions at the alpha(1)beta(2) intersubunit contacts are propagated to the unliganded heme in a manner that increases its affinity for oxygen. This effect contrasts sharply with the well-known reduction in oxygen affinity arising from these same contacts when unliganded dimers are assembled to form unliganded alpha(2)beta(2) tetramers. The magnitude of the enhancement in affinity at the unliganded site in triliganded tetramers (0.81 kcal, 3.39 kJ) is approximately one-fourth as great as the reduction in affinity on each heme site that arises from subunit assembly of the unliganded tetramer. The terms "quaternary constraint" and "quaternary enhancement" are employed to describe these oppositely directed effects of intersubunit interaction upon heme-site affinity. Experimental results also suggest that dimers bind oxygen with a higher affinity than monomeric alpha and beta chains do under the same temperature and buffer conditions (21.5 degrees C, pH 7.40, 0.1 M Tris.Hcl/0.1 M NaCl/1 mM Na(2)EDTA). Thus quaternary enhancement may be manifested at the alpha(1)beta(1) contacts. Implications of these results for models of the cooperative mechanism are discussed.


Reconstitution of membranes and embryonic development in dissociated blastula cells of the sea urchin by reinsertion of aggregation-promoting membrane proteins extracted with butanol. Blastula embryos of the sea urchin Paracentrotus lividus, when dissociated into single cells by exposure to Ca2+- and Mg2+-free sea water, reassociate spontaneously to form aggregates capable of development to the final larval form (pluteus). This aggregation is prevented by Fab fragments obtained by immunization with purified membranes from blastula embryos. The inhibition was reversed by soluble proteins extracted with butanol from purified membranes or from intact cells. These extracts also strongly stimulated the rate of reaggregation of dissociated cells in the absence of Fab fragments. Exposure of dissociated cells to 2.5% (vol/vol) butanol removed completely the protein(s) responsible for reaggregation of the cells without impairing their viability. Reaggregation and embryonic development were completely restored to the extracted cells by readdition of the proteins extracted from either membranes or cells. Extracted cells from Paracentrotus could be reconstituted with proteins from Arbacia.


Photobilirubin: an early bilirubin photoproduct detected by absorbance difference spectroscopy. Absorption of light converts bilirubin-IXalpha in solution to a mixture of what are probably cis-trans geometric isomers. This reaction is much faster than other photochemical reactions of bilirubin and reaches photoequilibrium before losses due to photooxidation are significant. At room temperature in the dark in the presence of trifluoroacetic acid or iodine or simply on standing, the photoproducts revert to the natural isomer. They also revert under visible light. Their formation and reversion can be followed by chromatography on polyamide and by absorbance difference spectroscopy.


Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins. Microtubule protein from HeLa cell extracts was purified by multiple cycles of polymerization and depolymerization in the absence of glycerol or other exogenous polymerization-stimulatory agents. Approximately 4-5% of the extract protein was tubulin, of which more than one-half was competent to participate in polymerization-depolymerization cycles. The purified HeLa microtubule protein preparations contained 95% tubulin after the second cycle of polymerization and depolymerization. Additional protein species bound specifically to and copurified quantitatively with microtubules throughout at least four cycles of polymerization and depolymerization. These microtubule-associated proteins (MAPs) were separated from tubulin by DEAE column chromatography. When added to purified brain or HeLa tubulin, these MAPs stimulated the polymerization of microtubules as assayed by electron microscopy and a quantitative sedimentation assay. The most prominent HeLa MAPs had molecular weights of approximately 210,000 and 120,000.


mRNA-directed biosynthesis of alpha subunit of thyrotropin: translation in cell-free and whole-cell systems. mRNA from mouse thyrotropic pituitary tumors was translated in frog oocytes (a whole-cell system) and in wheat germ extract and reticulocyte lysate (cell-free systems) in the presence of [(35)S]methionine. Synthesized peptides related to thyrotropin were identified in the three systems by immunoprecipitation with subunit-specific antisera developed against the alpha subunit of ovine lutropin (luteinizing hormone) and the beta subunit of bovine thyrotropin. In wheat germ extract and reticulocyte lysate, a single immunoprecipitable form of the alpha subunit of thyrotropin was synthesized with an apparent molecular weight of 14,000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the frog oocyte, three forms of immunoprecipitable alpha subunit of thyrotropin were synthesized with apparent molecular weights of 20,000, 14,000, and 10,000. The 20,000 form is similar to unlabeled rat pituitary standard alpha subunit and (35)S-labeled mouse tumor alpha subunit in cell cultures (20,000-21,000); thus, it may represent a precursor-cleaved and glycosylated form. The 14,000 form synthesized in all three systems probably represents the pre-alpha subunit of thyrotropin; the 10,000 form, synthesized only in the frog oocyte, could be a proteolytically cleaved but unglycosylated form. Because only the alpha subunit of thyrotropin was identified and no larger molecular weight immunoprecipitable form of either subunit was detected in any of the translation systems, alpha and beta subunits of thyrotropin appear to be translated from separate mRNAs.


Interaction of wild-type and variant mouse 3T3 cells with lectins from Bandeiraea simplicifolia seeds. An isolectin (BS I-B4) derived from Bandeiraea simplicifolia seeds and specific for terminal alpha-D-galactopyranosyl groups was found to be cytotoxic to Swiss 3T3 mouse cells. After mutagenesis and selection with BS I-B4, a variant clonal cell line resistant to both this isolectin and the alpha-D- and beta-D-galactose-binding lectin abrin was isolated. The parental cell line showed homogeneous and noninteracting binding sites for BS I-B4, whereas the variant cells exhibited a curved plot with a reduced number of binding regions. Another lectin, BS II, which is derived from the same seeds by specific for terminal N-acetyl-D-glucosaminyl groups, was cytotoxic to the variant but not the parental cells. These results suggest a possible lesion in the biosynthesis of cell surface structures resulting in the exposure of subterminal N-acetyl-D-glucosaminyl moieties in the variant line.


Procollagen segment-long-spacing crystallites: their role in collagen fibrillogenesis. Naturally occurring segment-long-spacing crystallites of procollagen collagen have been found in the culture medium of fibroblasts from chick embryo tendon, human skin, and dermatosparaxic calf skin; in whole-mount preparations of cultured human skin fibroblasts; in homogenates of lathyritic chick embryo tendon, cartilage, and cornea; and in a partially purified preparation of procollagen. Bundles of similar aggregates occurred within secretory vacuoles of collagen-synthesizing fibroblasts and chondrocytes. These observations suggest that fibroblasts and chondrocytes secrete procollagen assemblies that are stable in the extracellular environment. We propose that subsequent enzymatic processing is accompanied by direct incorporation of such structures into the assembling fibril, which then may be considered as an n X 67 nm staggered array of segment-long-spacing crystallites.


Conservative segregation of parental histones during replication in the presence of cycloheximide. Long stretches of protein-free, nonbeaded DNA were observed electron microscopically in nuclear spreads prepared from cells that had replicated their DNA in the absence of protein synthesis. The amount of this DNA increased with increasing time of replication in the presence of cycloheximide and was greatly decreased when replication was inhibited with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside). This DNA is considered to be "free" DNA because it has the same diameter as marker PM2 DNA and it is preferentially sensitive to DNase I digestion. Reversal of the cycloheximide block resulted in a burst of histone synthesis and repair of the depleted chromatin within 5 min. In addition, 26 presumptive replication forks were observed with beaded chromatin on two arms and free DNA on the third. These results suggest that new histones are usually deposited onto new DNA, that the cellular histone pool is very small, that histone migration is minimal in vivo for at least 18 hr, that for most fibers nuclesome assembly and segregation is conservative for stretches of DNA as long as 100 kbases, and that some part of the octameric histone core may remain bound to DNA during the replication process. The regularity we have observed for the assembly and segregation of nucleosomes is likely to be important for our understanding of how chromosomal information is segregated during development.


Resonance Raman studies of bathorhodopsin: evidence for a protonated Schiff base linkage. A dual beam pump/probe technique has been used with a 585-nm probe wavelength to obtain maximal resonance enhancement of the Raman lines of bathorhodopsin in a photostationary steady-state mixture at -160 degrees C. These studies show that bathorhodopsin has a protonated Schiff base vibration at 1657 cm(-1) which shifts upon deuteration to 1625 cm(-1). Within our experimental error (+/-2 cm(-1)) these frequencies are identical to those observed in rhodopsin and isorhodopsin. These effects show that the strength of the C=N bond and the degree of protonation of the Schiff base nitrogen are the same in bathorhodopsin, rhodopsin, and isorhodopsin. The implication of these results for the structure of the retinal chromophore in bathorhodopsin are discussed. The resonance Raman spectrum of pure bathorhodopsin has been generated by accurately subtracting the residual contributions of rhodopsin and isorhodopsin from spectra of the low temperature photostationary mixture. Bathorhodopsin is found to have lines at 853, 875, 920, 1006, 1166, 1210, 1278, 1323, 1536, and 1657 cm(-1). Also, by using an intensified vidicon detector, we have observed Raman scattering from bathorhodopsin at room temperature by generating a photostationary steady state with pulsed laser excitation. At room temperature the three characteristic lines of bathorhodopsin are found at 858, 873, and 920 cm(-1). The fact that the frequencies of these bathorhodopsin lines are nearly identical at both temperatures implies that the retinal conformation in bathorhodopsin formed at -160 degrees C is the same as that formed at room temperature.


High affinity binding of chylomicron remnants to rat liver plasma membranes. The binding of chylomicron remnants rat liver plasma membranes was studied. Liver membranes bound up to 8 times more remnants than they bound chylomicrons. The remnant particle appeared to bind to the membrane as a unit. Remnant binding was greatest to liver plasma membrane; only one third as much binding was observed with whole liver homogenate, and virtually no binding occurred to erythrocyte membranes or glass. Binding was saturable and had kinetics compatible with the existence of a high affinity site. Half-maximal binding occurred at 27 micron. Competitive binding studies revealed no competition with albumin, a triglyceride dispersion, cholesterol/lecithin vesicles, very low density lipoprotein, or low density lipoprotein. Some displacement of remnant binding was observed with chylomicrons and high density lipoprotein. Binding was decreased by treatment of the membranes with trypsin or the presence of heparin in the incubation medium. These studies suggest that there is a high affinity receptor for the chylomicron remnant on the surface of the hepatocyte.


Glycoprotein, elastin, and collagen secretion by rat smooth muscle cells. Smooth muscle cells from rat heart secreted extracellular matrix components at high rates for many generations in culture. The matrix proteins remained anchored to the culture dish and were characterized after removal of cellular material with sodium dodecyl sulfate. Sequential enzyme digestion demonstrated the presence of at least three components, including glycoprotein(s), elastin, and collagen. Prolonged extraction of the matrix with detergent under reducing conditions solubilized a fucosylated glycoprotein having an apparent molecular weight of 250,000 and two other proteins with molecular weights of 72,000 and 45,000, respectively. Sublines derived from discrete colonies of smooth muscle cells synthesized all of the matrix components, and the proportion of collagen secreted by some sublines increased with time in culture. The biosynthesis of a mixed extracellular matrix and the relationships among the component proteins were therefore studied in one system producing milligram quantities of material.


Relationship between RNA content and progression of lymphocytes through S phase of cell cycle. A flow cytometric technique for simultaneous measurements of RNA and DNA in individual cells has been applied to correlate the content of cellular RNA with the rate of progression of cells through the S phase. Human peripheral lymphocytes stimulated with phytohemagglutinin were blocked at the G1/S phase boundary by hydroxyurea or 5-fluorodeoxy-uridine treatment. Cells in the G1 phase as well as cells blocked at the G1/S phase boundary showed high heterogeneity with respect to stainable RNA content. After release from the block, the cells traversed the S phase at rates proportional to the quantity of stainable RNA per cell. Cells with the highest RNA content completed DNA replication 5 hr after release from the block; the cells with minimal RNA traversed the S phase at one-fifth of this rate. The large intercellular variation in stainable RNA and length of the S phase may be due to functional heterogeneity in the lymphocyte population. Our results suggest a correlation between the number of ribosomes and the rate of DNA replication in lymphocytes.


Monoclonal antibodies reacting with murine teratocarcinoma cells. Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000.


Evolutionary change in 5S RNA secondary structure and a phylogenic tree of 54 5S RNA species. Secondary structure models of 54 5S RNA species are constructed based on the comparative analyses of their primary structure. All 5S RNAs examined have essentially the same secondary structure. However, there are revealing characteristic differences between eukaryotic and prokaryotic types. The prokaryotic 5S RNAs may be further classified into two types, one having 120 nucleotides (120-N type) and another having 116 (116-N type). A possible mechanism for the conversion of the prokaryotic 116-N type to the 120-N type 5S RNAs (or vice versa) is discussed on the basis of their nucleotide alignments. Finally, by comparing the nucleotide alignments, we propose a phylogenic tree of the 54 5S RNA species.


Genetic translocation in Staphylococcus aureus. A 5.2-kilobase pair transposon, Tn551, has been found in Staphylococcus aureus, a Gram-positive bacterium. Initially detected on plasmid pI258, it undergoes rec-independent transposition to multiple chromosomal and plasmid sites, sometimes causing insertional inactivation. Unlike most other transposons, Tn551 undergoes apparently precise excision as a rule. The initial observation of Tn551 transition involved UV inactivation of the carrier plasmid; this would appear to be a general means of detecting transposable elements.


Genetic control of cobalamin binding in normal and mutant cells: assignment of the gene for 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase to human chromosome 1. When extracts prepared from cultured human or rodent fibroblasts grown in medium containing [(57)Co]cobalamin were analyzed by polyacrylamide gel electrophoresis, most of the intracellular radioactivity migrated with the activity of the cobalamin-dependent enzyme 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase (EC 2.1.1.13). Because the rodent and human forms of this enzyme are electrophoretically different, we used the binding of [(57)Co]cobalamin to detect the presence of the human methyltransferase isozyme in rodent-human somatic cell hybrids. As expected, binding and methyltransferase activities were found to cosegregate, thus confirming genetically their electrophoretic identity. Accordingly, we examined the [(57)Co]cobalamin-binding patterns and human chromosome contents of a panel of 12 rodent-human hybrid clones, and concluded that the gene for the methyltransferase (designated Mtr) is located on human chromosome 1. Using this information, we probed the nature of the molecular defect exhibited by fibroblasts cultured from patients expressing the cbl C mutation. Although these cells are unable to associate newly taken up [(57)Co]cobalamin with the methyltransferase, hybrids of mouse L-cells and cbl C cells containing chromosome 1 show a "reappearance" of the human [(57)Co]cobalamin-methyltransferase. These results indicate that the cbl C mutation does not affect the methyltransferase apoprotein, but rather some metabolic step that must convert cobalamin to a chemical form capable of attaching to the enzyme.


Emetine resistance in Chinese hamster cells is linked genetically with an altered 40S ribosomal subunit protein, S20. Emr-2 is an emetine-resistant (Emr) Chinese hamster ovary cell mutant that contains a single electrophoretically altered ribosomal protein, a component of its 40S ribosomal subunit [Boersma, D., McGill, S., Mollenkamp, J. & Roufa, D.J. (1979) J. Biol. Chem. 254, in press]. This report describes a genetic experiment designed to test linkage between the genes that specify the altered ribosomal protein, S20*, and emetine resistance in Emr clones that segregated from cultures of Emr-2 cells hybridized with emetine-sensitive cells. The data described indicate that Emr and S20* phenotypes are due to mutations linked to the same chromosome in the Chinese hamster genome; most likely they are due to the same mutation. The data also confirm earlier speculations by others that the Emr locus in Chinese hamster cells is hemizygous.


Evolutionary effects of contagious and familial transmission. Two models involving non-Mendelian transmission of a discrete valued trait through within- and across-generation contagion are proposed in an investigation of the joint evolution of phenotype and genotype. A single locus with two alleles determines susceptibility to contagion. The incorporation of within-generation contagious transmission extends the parameter ranges allowing phenotypic polymorphism and introduces a new phenotypic equilibrium configuration. The latter is characterized by a threshold in the initial value of the trait which determines whether the trait can increase. Phenotypic evolution is accelerated by within-generation contagion, but the rate of genetic evolution is retarded relative to that under uniparental transmission across generations. The second model studied allows the trait to be acquired, at genotype-dependent rates, even if the transmitting parent does not have the trait. Both the pattern of phenotypic transmission and the selection on the trait influence the course of evolution. Some important aspects of the structure of the one locus-two allele model are shown to be preserved with more alleles. At equilibrium, the leading eigenvalue of the transmission-selection matrix assumes the role of genotypic fitness.


Identification of a non-H-2 gene (Rfv-3) influencing recovery from viremia and leukemia induced by Friend virus complex. The dominant C57BL/10 allele of a single autosomal, non-H-2 gene (Rfv-3) was found to be required for recovery from viremia and leukemia induced by Friend virus complex in H-2b/b mice. In H-2a/a mice, the Rfv-3 gene apparently influenced recovery from viremia in the presence of persistent leukemia because these mice lacked the appropriate H-2 genotype for recovery from leukemia. The Rfv-3 gene was distinct from the Fv-2 gene because recovery from viremia was seen in recombinant-inbred mice with the Fv-2s/s genotype. Furthermore, backcross studies indicated that Rfv-3 and Fv-2 were not linked. The Rfv-3 gene may act by influencing the specific anti-FV humoral antibody response.


Genetic lesions induced by chemicals in spermatozoa and spermatids of mice are repaired in the egg. Conclusive proof that the mouse egg is capable of carrying out repair of genetic lesions present in the male genome was obtained through dominant-lethal studies of chemically treated spermatozoa and spermatids and through cytological analysis of first-cleavage metaphases. The maximum difference in repair capability between stocks of females, found for isopropyl methanesulfonate treatment, was large; considerably smaller differences were found for ethyl methanesulfonate, triethylenemelamine, and benzo[a]pyrene treatments; and no difference was found for x-ray treatment.


Another approach to in vivo estimation of genetic damage in humans. Direct measurements of the genetic hazards to man have been determined previously by the appearance of elevated in vivo levels of chromosome aberrations. Here we report that interindividual variations in N-acetoxy-2-acetyl-aminofluorene-induced unscheduled DNA synthesis values parallel the actual in vivo levels of chromosome damage and, therefore, may be a rapid useful supplement in genetic risk assessment for humans.


Incorporation of glucocerebrosidase into Gaucher's disease monocytes in vitro. Several carriers were evaluated for use in the delivery of exogenous glucocerebrosidase to monocytes from Gaucher's disease patients. Only gamma globulin-coated, resealed erythrocytes proved to be an effective vehicle for enzyme delivery. Glucocerebrosidase added in this manner normalized intracellular enzyme levels for at least 18 hr. In this model system for the study of enzyme replacement therapy, soluble enzyme, enzyme in uncoated resealed erythrocytes, and enzyme incorporated into liposomes were ineffective.


Modification of the blood-brain barrier: increased concentration and fate of enzymes entering the brain. The blood-brain barrier of rats was opened reversibly by infusing a hyperosmotic solution of arabinose into the external carotid artery. Permeability was increased maximally in the first 15 min and remained slightly elevated at 1 hr. Osmotic barrier opening significantly increased brain uptake of intravenously injected alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.2.4) (derived from human placenta) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). By injection of 4 X 10(5) units of alpha-mannosidase into an animal, brain activity rose to about twice the normal control activity of the enzyme. After 30 min, activity of administered enzyme in the extracellular space of the brain was calculated to be 30% of the serum concentration. Biochemical and histological studies with horseradish peroxidase showed that exogenously administered enzyme entered the brain extracellular space immediately after barrier opening and was incorporated within neuronal lysosomal packets during the next 24 hr. Measurable peroxidase activity was found in brain as much as 72 hr after osmotic treatment. The results demonstrate that the blood-brain barrier can be reversibly opened to enzymes, that a glycoprotein enzyme is incorporated into neuronal lysosomes, and that the brain may now be considered a potential target for enzyme replacement therapy in heritable metabolic disorders.


Metal-binding and detoxification effect of synthetic oligopeptides containing three cysteinyl residues. Metallothionein is a naturally occurring metal-binding protein with high cysteine content. Oligopeptides containing three cysteinyl residues and having amino acid sequences analogous to portions of this protein were synthesized by the solid-phase method. Strong affinity of the synthetic peptides to Cd2+ and Zn2+ was observed, and the dissociation constants of the peptide-metal complexes were 2-4 orders of magnitude lower than those of cysteine-metal and dithioerythritol-metal complexes. Effectiveness of detoxification of the peptides against Cd toxicity was demonstrated by the higher survival rates of mice treated with the peptides and by the neutralization of Cd toxicity by the peptides in tissue cultures.


Humoral factor that specifically regulates prothrombin (factor II) levels (coagulopoietin-II). Prothrombin (Factor II) was decreased in rabbits by injection of a goat antiserum to prothrombin. When plasma from these rabbits was injected into normal animals, circulating prothrombin increased in the recipients, suggesting transfer of a humoral regulating factor ("coagulopoietin-II"). This coagulopoietin-II resisted boiling for 20 min. On Sephadex G-50 gel filtration, it remained in the retained volume, suggesting a molecular weight of less than 30,000. In an in vitro liver system, prothrombin increased in the incubation medium in the presence of coagulopoietin-II.


Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by Methanosarcina barkeri. A number of N-methyl compounds, including several methylamines, creatine, sarcosine, choline, and betaine, were readily fermented by enrichment cultures yielding methane as a major product. Methylamine, dimethylamine, trimethylamine, and ethyldimethylamine were fermented by pure cultures of Methanosarcina barkeri; except for ethyldimethylamine, these amines are considered important substrates of this methanogenic microorganism. Creatine, sarcosine, choline, and betaine were fermented to methane only by mixed cultures. During growth of M. barkeri on methyl-, dimethyl-, or trimethylamine, methanol was not excreted into the medium. The fermentation of trimethylamine gave rise to an intermediary accumulation of methyl- and dimethylamine in the medium. An accumulation of methylamine during the fermentation of dimethylamine was not observed. Methane and ammonia were produced from the three methylamines by M. barkeri in amounts expected on the basis of the appropriate fermentation equations. The growth yield was 5.8 mg of cells (dry weight) per mmol of methane and was not dependent on the kind of methyl compound used as substrate.


Distribution of binding sites for 125I-labeled alpha-bungarotoxin in normal and deafferented antennal lobes of Manduca sexta. 125I-Labeled alpha-bungarotoxin has been used to determine the distribution of putative acetylcholine receptors in normal and chronically deafferented antennal lobes in the brain of the moth Manduca sexta. Toxin-binding sites are confined to synaptic regions in deafferented lobes. These findings suggest that receptors can develop in the insect central nervous system independently of normal synaptic influences.


Complementary carrier peptide synthesis: general strategy and implications for prebiotic origin of peptide synthesis. A method for peptide synthesis is proposed based on a template-directed scheme that parallels that of the native ribosomal mechanism. In this procedure, peptide bond formation is facilitated by the juxtaposition of aminoacyl and peptidyl oligonucleotide carriers bound adjacent to one another on an oligonucleotide template. The general strategy of the synthesis and relevant model studies are described. The scheme provides an intrinsic mechanism by which oligonucleotides can direct the synthesis of polypeptides in the absence of protein or ribosomal machinery and, as such, suggests a model for the origin of prebiotic protein synthesis.


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


Prolyl-leucyl-glycinamide, cyclo(leucylglycine), and derivatives block development of physical dependence on morphine in mice. Pro-Leu-Gly-NH2 (MIF) and several structural analogues, all injected in 50-microgram doses daily in mice receiving morphine chronically, were found to prevent development of physical dependence as measured by changes in body temperature associated with naloxone-induced withdrawal. Dose-response studies, using again a protocol of daily injections of peptide at 50, 5, 0.5, 0.05, 0.005 microgram per mouse revealed MIF and cyclo(Leu-Gly) to be the most potent peptides and to be effective in blocking physical dependence to morphine at a dose as low as 0.5 and 0.05 microgram per mouse, respectively. The benzyloxycarbonyl derivative of MIF, Pro-Leu, and Pro- -Leu exhibited significant activities down to a dose of 5 microgram of peptide per mouse.


Cholecystokinin octapeptide-like immunoreactivity: histochemical localization in rat brain. Cholecystokinin octapeptide-like (CCK-OP-like) immunoreactivity was localized in the rat brain by using the indirect immunofluorescence method. Specificity in immunohistochemical studies was demonstrated by the virtual elimination of staining with either preimmune sera or sera preadsorbed with CCK-OP and by the achievement of similar fluorescent patterns with two different primary anti-CCK-OP sera. CCK-OP-like fluorescence was localized in neuronal cell bodies, fibers, and varicose terminals. The most dense collections of CCK-OP cells occurred in the periaqueductal gray and in the dorsomedial hypothalamus. Substantial numbers of cells and fibers also were present in the medial/dorsal and perirhinal cortex; more limited groups of cells were found in the pyramidal layer of the hippocampus and in the dorsal raphe.


Tetraethylammonium: voltage-dependent action on endplate conductance and inhibition of ligand binding to postsynaptic proteins. Tetraethylammonium (Et(4)N(+)) ions depressed the amplitude and accelerated the decay rate of spontaneously occurring and nerve-evoked endplate currents (EPCs) in frog sartorius muscle. The relationship between peak EPC amplitude and membrane potential became nonlinear in the presence of 100 muM Et(4)N(+), and with drug concentrations of 250 muM or greater the current-voltage relationship exhibited negative conductance in the hyperpolarized region. Et(4)N(+) modified the exponential dependence of the EPC decay on membrane potential such that the decays between -150 and -50 mV were abbreviated and voltage independent but remained near control levels at more positive membrane potentials. The minimal effective concentration of Et(4)N(+) for altering the EPC time course was 10, and maximal effects were attained with 100 muM. Little additional shortening in the EPC decay phase was detected on raising the drug concentration to 1000 muM. Acetylcholine noise analysis revealed a voltage-dependent reduction in the mean channel open time, which was comparable in magnitude to the shortening in the EPC decay, and a depression of single-channel conductance. In concomitant biochemical studies, Et(4)N(+) was found to inhibit the binding of both [(3)H]acetylcholine and [(3)H]perhydrohistrionicotoxin to receptor-rich membranes from the electric organ of Torpedo ocellata with K(i) values of 200 muM and 280 muM, respectively. These results suggest that Et(4)N(+) interacts with both the acetylcholine receptor and its associated ionic channel. The voltage-dependent actions of Et(4)N(+) are attributed to blockade of the ionic channel in closed as well as open conformation.


Mouse mammary epithelial cells on floating collagen gels: transepithelial ion transport and effects of prolactin. Epithelial cells dissociated from midpregnant BALB/c mouse mammary glands were cultured for as long as 20 days as confluent monolayers on floating collagen gels. Detached gels bearing monolayers were placed in lucite Ussing chàmbers for measurement of transepithelial potential difference (PD), short-circuit current (I(sc)), resistance (R), and unidirectional fluxes of Na(+) and Cl(-) during short-circuit current conditions (PD = 0). With Hanks' solution bathing both sides of cultures maintained with insulin and cortisol, PD = -12.8 mV (serosal side ground), I(sc) = 24.6 muA/cm(2), and R = 507 omega.cm(2). Net absorption of Na(+) equaled I(sc), and there was no net Cl(-) transport. PD and I(sc) were reduced 50% by mucosal addition of 10 muM amiloride and to zero by metabolic inhibition with nitrogen gas or by serosal addition of 0.1 mM ouabain. In similar cultures supplemented with prolactin, PD and I(sc) increased to -15.8 mV and 48.0 muA/cm(2), respectively, and R decreased to 374 omega.cm(2). Inhibitor effects were similar to those seen in prolactin-free cultures. Prolactin exposure resulted in a 3-fold increase in net absorption of Na(+). Na(+) absorption was not equivalent to I(sc), and there was little Cl(-) absorption; therefore, prolactin induced active transport of other, as yet unidentified, ions. These effects of prolactin require at least 3 days to occur and cannot be attributed to the known contamination with neurohypophysial hormones. The prolactin-induced increase in Na(+) absorption parallels its Na(+)-retaining ability in lower vertebrates and could be part of the mechanism that keeps milk Na(+) concentration low in intact glands.


Subnanosecond motions of tryptophan residues in proteins. The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (varphi) of 9.9 nsec at 20 degrees C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits varphi of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8 degrees C (varphi = 31.4 nsec), whereas at 43 degrees C it rotates rapidly (varphi(1) = 0.14 nsec) within a cone of semiangle 26 degrees in addition to rotating together with the whole protein (varphi(2) = 14 nsec). Of particular interest in the large angular range (semiangle, 34 degrees ) and fast rate (varphi(1) = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.


Chicken lysozyme gene contains several intervening sequences. The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.


Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog. Procaine amide azide, a derivative of the local anesthetic procaine amide, was prepared and its interactions with acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax were studied. Procaine amide azide was radiolabeled by a method that may be of general use for the preparation of other radioactive tertiary amines. When low concentrations of 3H-labeled procaine amide azide were photolyzed in the presence of receptor-rich membrane preparations, a simple pattern of incorporated radioactivity was seen after electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. Only two major labeled bands were seen, corresponding to apparent Mr of about 43,000 and 90,000. When the length of the gels was increased, the labeled band of lower Mr was resolved into two labeled proteins, one major and one minor, with apparent Mr of 43,000 and 40,000, respectively. The radioactivity incorporated into the protein of Mr 40,000 could be attributed to interaction of [3H]procaine amide azide with cholinergic ligand-binding sites, whereas labeling of the polypeptide of Mr 43,000 appears to represent interaction of the photolabile derivative with another class of sites. The labeling component of 90,000 Mr could be removed by preparation of membrane fragments in iodoacetamide-containing buffer and therefore appeared unrelated to the acetylcholine receptor.


Competitive inhibition of colchicine binding to tubulin by microtubule-associated proteins. Microtubule-associated proteins (MAPs) promote tubulin polymerization, whereas colchicine inhibits this process. In this paper, MAPs have been shown to inhibit colchicine binding to tubulin in a competitive manner. Attempts were made to identify which of the MAPs fraction(s) was responsible; both tau protein (a thermostable molecule with a molecular weight of approximately 70,000) and a high molecular weight fraction (HMW) were able to compete with colchicine. In contrast, Mg2+, which also induces microtubule assembly in vitro, had no effect on colchicine binding to tubulin.


Stimulation of benzodiazepine receptor binding by gamma-aminobutyric acid. The effect of the neurotransmitter gamma-aminobutyric acid (GABA) on high-affinity binding of benzodiazepines to brain membranes has been investigated. GABA stimulated [3H]diazepam binding by more than 100% when extensively washed membranes from brain tissue were used. This GABA-stimulated benzodiazepine binding occurred in all brain regions examined. The stimulation was specific for GABA agonist. It was inhibited by the GABA receptor blocker bicuculline methiodide. A large number of compounds structurally closely related to GABA but without direct effect on the GABA receptor failed to enhance [3H]diazepam binding. The stimulation of benzodiazepine binding was caused by an increase in affinity; the number of binding sites remained unchanged. Half-maximal activation of [3H]diazepam binding occurred in the presence of 300 nM muscimol or 900 nM GABA. beta-Guanidinopropionic acid and imidazoleacetic acid were much weaker activators. It is suggested that the described stimulation of benzodiazepine high-affinity binding is mediated by a receptor for GABA. This site of GABA action exhibits different properties when compared to GABA receptors, as characterized by high-affinity binding of GABA agonists.


Absence of heme-localized strain in T state hemoglobin: insensitivity of heme-imidazole resonance Raman frequencies to quaternary structure. Substitution of pentadeuterated 2-methylimidazole in (2-methylimidazole)-Fe(II)-protoporphyrin IX, a model complex for deoxyHb, shifts three bands in the low-frequency resonance Raman spectrum 380 leads to 373 cm-1, 348 leads to 345 cm-1, and 220 leads to 218 cm-1. The first of these is assigned primarily to Fe-imidazole stretching, and the other two are assigned to porphyrin deformation modes with substantial Fe-pyrrole stretching contributions. The three bands are observed in deoxyHb and Mb. The Fe-pyrrole modes are at essentially the same frequencies in the two proteins, but the Fe-imidazole mode is 6 cm-1 lower in deoxyHb than Mb, implying a slight alteration in the heme-imidazole linkage. No change greater than 2 cm-1 is observed when Hb Kempsey is switched from the R to the T state. This observation places an upper limit on the energy stored in the Fe-imidazole bond of T state deoxyHb, which is estimated to be less than 0.2 kcal/mol (less than 836.8 J/mol).


Conformation of nucleosides: circular dichroism study on the syn-anti conformational equilibrium of 2-substituted benzimidazole nucleosides. The solution conformations of 2-substituted derivatives of 1-(beta-D-ribofuranosyl)benzimidazole have been determined by circular dichroism spectroscopy in aqueous solutions. It is shown that analogs with methyl, amino, or methylamino substituents at position 2 of the benzimidazole ring (position 8 of the purine ring) have predominantly anti conformations, whereas analogs with chloro, aza, methoxy, or methylmercapto substituents have predominantly syn conformations. The preferred solution conformations of the benzimidazole nucleosides and analogous purine nucleosides are compared. The results demonstrate that the replacement of nitrogen by carbon at position 3 of the purine ring of purine (beta) nucleosides leads to important conformational consequences, which are strengthened or neutralized by substituents at position 8 of the purine ring.


Do cleavages of amides by serine proteases occur through a stepwise pathway involving tetrahedral intermediates? The mechanism of the serine protease-catalyzed cleavage of amides (acylation) was examined in terms of the basicity of the functional groups participating in the catalysis. It is proposed that the reaction does not proceed through a stepwise pathway, as opposed to the cleavage of esters and anilides, which start with general base-catalyzed formation of the tetrahedral intermediate followed by its general acid-catalyzed breakdown. Instead, the proton abstracted from the hydroxyl group of the serine by the imidazolyl group of the histidine is donated to the nitrogen atom of the leaving group of the amide before the bond between the carbonyl carbon atom of the amide and the attacking serine oxygen atom is completed. Reactions proceed by a SN2-like reaction through the cooperation of acid catalysis by the imidazolyl cation and nucleophilic attack by the serine. The mechanisms of the enzymatic hydrolyses of anilides and esters proceed through a discrete tetrahedral intermediate, but the enzymatic hydrolyses of amides probably do not.


Amino-acid sequence of Tetrahymena histone H4 differs from that of higher eukaryotes. A partial amino-acid sequence of Tetrahymena histone H4 has been determined and differs significantly from the sequence of calf or pea H4. The amino terminus of Tetrahymena H4, unlike that of other H4s so far examined, is not acetylated. Of 66 residues determined, one is a single-residue insertion, one a single-residue deletion, and thirteen are amino-acid replacements with respect to the calf thymus H4 sequence. Most of the amino-acid replacements are nonconservative and are distributed nonrandomly, with a strong concentration in the amino-terminal arm. The first four lysines are partially acetylated. All but two of the replacements can be explained by single nucleotide substitutions at the level of the gene. The similarity in sequence of calf and pea H4 coupled with the substantial differences displayed  by Tetrahymena H4 suggest that the divergence of protozoa and higher eukaryotes substantially antedates the divergence of plants and animals. Furthermore, quantitative analysis of the data requires either that the rate of H4 evolution be considerably more rapid than previously thought or that the rate be different for different periods of evolution or for different lines of descent.


Sodium-calcium ion exchange in cardiac membrane vesicles. Membrane vesicles isolated from rabbit ventricular tissue rapidly accumulated Ca2+ when an outwardly directed Na+ gradient was formed across the vesicle membrane. Vesicles loaded internally with K+ showed only 10% of the Ca2+ uptake activity observed with Na+-loaded vesicles. Dissipation of the Na+ gradient with the monovalent cation exchange ionophores nigericin or narasin caused a rapid decline in Ca2+ uptake activity. The Ca2+-ionophore A23187 inhibited Ca2+ uptake by Na+-loaded vesicles and enhanced the rate of Ca2+ loss from the vesicles after uptake. Efflux of preaccumulated Ca2+ from the vesicles was stimulated 30-fold by the presence of 50 mM Na+ in the external medium. Na+-dependent uptake and efflux of Ca2+ were both inhibited by La3+. The results indicate that cardiac membrane vesicles exhibit Na+-Ca2+ exchange activity. Fractionation of the vesicles by density gradient centrifugation revealed a close correspondence between Na+-Ca2+ exchange activity and specific ouabain-binding activity among the various fractions. This relationship suggests that the observed Na+-Ca2+ exchange activity derives from the sarcolemmal membranes within the vesicle preparation.


Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eIF-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. The phosphorylation of purified protein synthesis factors catalyzed by protein kinase preparations isolated from interferon-treated human amnion cells was examined. Ribosomal salt-wash fractions prepared from interferon-treated human cells contained a protein kinase that catalyzed the [gamma-(32)P]ATP-mediated phosphorylation of the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2alpha); this kinase activity was significantly enhanced in interferon-treated as compared to untreated cells. The tryptic [(32)P]phosphopeptide pattern obtained for eIF-2alpha phosphorylated by the interferon-mediated human kinase was indistinguishable from the pattern obtained for eIF-2alpha phosphorylated by the hemin-regulated rabbit reticulocyte kinase when analyzed by thin-layer chromatography with three different solvent systems and by high-voltage electrophoresis. O-[(32)P]Phosphoserine was liberated by partial acid hydrolysis from eIF-2alpha phosphorylated by either the human or the rabbit kinase. In addition to the phosphorylation of eIF-2alpha, interferon treatment of human cells enhanced the phosphorylation of two additional ribosome-associated proteins designated P(1) and P(f). The major phosphoester linkage observed for the human, as well as murine, phosphoprotein P(1) was O-phosphoserine. The interferon-mediated phosphorylation of both eIF-2alpha and protein P(1) was dependent upon the presence of RNA with double-stranded character; P(f) phosphorylation was not affected by double-stranded RNA. These results suggest that the interferon-mediated ribosome-associated human protein kinase catalyzes the phosphorylation of eIF-2alpha in a site-specific manner that is apparently identical with the reaction catalyzed by the hemin-regulated rabbit reticulocyte kinase; hence, the phosphorylation of eIF-2 may play a role in regulating the initiation of translation in interferon-treated cells.


Preparative and analytical purification of DNA from agarose. 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.


Effect of benzo[a]pyrene-diolepoxide on infectivity and in vitro translation of phage MS2 RNA. Previous studies have shown that alkylation of MS2 RNA by certain derivatives of polycyclic aromatic hydrocarbons renders it noninfectious. Since phage RNA serves as a template for translation and transcription, either of these RNA-directed processes, or both, could be responsible in vivo for the inhibition of phage replication by metabolically activated hydrocarbons. The present study correlates the degree of inhibition of MS2 RNA infectivity, at various levels of alkylation by (+/-)-trans, 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene, with the translation efficiency in vitro of the same alkylated RNA for the synthesis of viral synthetase and of maturation and coat proteins. The results indicate that dihydroxyepoxy-tetrahydrobenzo[a]pyrene modification of MS2 RNA impairs its template capacity for the synthesis of phage-specific proteins; this inhibition is insufficient, however, to account for the loss of RNA infectivity at lower molar ratios of alkylation. For the three viral proteins synthesized in vitro, the translation of RNA synthetase is much more sensitive to MS2 RNA modification than either coat or maturation protein synthesis. Our results also indicate that the loss of viral RNA infectivity follows a single-hit inactivation mechanism, whereas several alkylation events in the viral RNA synthetase cistron may be necessary to block translation of this gene product.


Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin. The globin gene is preferentially sensitive to digestion by DNase I in erythrocyte chromatin but not in brain, fibroblast, or oviduct chromatin. Elution of the erythrocyte chromatin with 0.35 M NaCl leads to no detectable change in the gross structure of individual nucleosomes; however, in this depleted chromatin the globin gene is no longer preferentially sensitive to DNase I. Reconstitution of the depleted chromatin with either the entire 0.35 M NaCl fraction or a subclass from this fraction greatly enriched in two high mobility group proteins (nos. 14 and 17) results in the successful reconstitution of DNase I sensitivity of the globin gene. For all of these preparations, the inactive ovalbumin gene exhibited no preferential sensitivity to DNase I. Reconstitution of the erythrocyte 0.35 M NaCl fraction with depleted brain chromatin resulted in no preferential sensitivity of the globin gene in brain chromatin; however, reconstitution of the brain 0.35 M NaCl fraction with depleted erythrocyte chromatin led to successful reconstitution of DNase I sensitivity of the globin gene. Thus, the eluted proteins responsible for conferring DNase I sensitivity are probably not tissue-specific and probably do not recognize specific DNA sequences.


Initial site of insulin cleavage by insulin protease. Exposure of insulin to insulin protease (insulinase, EC 3.4.22.11), a degradative enzyme with considerable specificity toward insulin, results in alterations in the properties of the insulin molecule. Limited degradation by the enzyme results in a decrease in the ability of insulin to bind to membrane receptors with less change in the immunoprecipitability or trichloracetic acid precipitability of the hormone. Limited degradation by insulin protease also alters insulin so that the molecule becomes susceptible to attack by nonspecific endopeptidases which have no effect on unaltered insulin. These data demonstrate the production of an intermediate in the proteolytic degradation of insulin. By labeling with [14C]dansyl chloride, an insulin intermediate with three amino-terminal residues, glycine, phenylalanine, and leucine, was identified. Analysis of this intermediate demonstrated that it was composed of an intact A chain and a B chain cleaved between residues B16 and B17, with the three peptide chains held together by disulfide bonds. Based on these findings, we hypothesize that a stepwise degradation of insulin occurs in vivo and that an early step in the process is the cleavage between B16 and B17 that renders the molecule sucseptible to further degradation by nonspecific proteases.


Human leukocyte interferon: production, purification to homogeneity, and initial characterization. A method of fractionating proteins by high-performance liquid partition chromatography has been developed and used for isolation and purification to homogeneity of one of the species of human leukocyte interferon. The homogeneous interferon exhibited a sharp peak on high-performance liquid chromatography and a single narrow band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Extraction of the gel gave a single sharp peak of antiviral activity coinciding with the protein band. The specific activity of pure interferon was found to be 2--4 X 10(8) units/mg, based on amino acid analysis. The molecular weight is 17,500--18,000.


Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions. Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [(3)H]tetraphenylphosphonium (TPP(+)) against a concentration gradient. The steady-state level of TPP(+) accumulation is about twice as great in physiological media of low K(+) concentration (i.e., 5 mM K(+)/135 mM Na(+)) than in a medium of high K(+) concentration (i.e., 121 mM K(+)/13.5 mM Na(+)). The latter manipulation depolarizes the NG108-15 plasma membrane and indicates that the resting membrane potential (DeltaPsi) is due primarily to a K(+) diffusion gradient (K(in) (+) --> K(out) (+)). TPP(+) accumulation is time and temperature dependent, achieving a steady state in 15-20 min at 37 degrees C, and is a linear function of cell number and TPP(+) concentration (i.e., the concentration gradient is constant). The difference in TPP(+) accumulation in low and high K(+) media under various conditions has been used to calculate mean (+/-SD) DeltaPsi values of -56 +/- 3, -63 +/- 4, and -66 +/- 5 mV at 26, 33, and 37 degrees C, respectively. Importantly, these values are virtually identical to those obtained by direct electrophysiological measurements made under the same conditions. TPP(+) accumulation is abolished by the protonophore carbonylcyanide-m-chlorophenylhydrazone, whereas the neurotoxic alkaloid veratridine diminishes uptake to the same level as that observed in high K(+) media. In addition, the effect of veratridine is dependent upon the presence of external Na(+) and is blocked by tetrodotoxin. The steady-state level of TPP(+) accumulation is enhanced by monensin, indicating that this ionophore induces hyperpolarization under appropriate conditions. Finally, ouabain has essentially no effect on the steady-state level of TPP(+) accumulation in short-term experiments, suggesting that Na(+),K(+)-ATPase activity makes little contribution to the resting potential in these cells. Because many of these observations are corroborated by intracellular recording techniques, it is concluded that TPP(+) distribution measurements can provide a biochemical method for determining membrane potentials in populations of cultured neuronal cells.


Adenovirus DNA replication in vitro.

Adenovirus DNA replication in vitro. A soluble extract from the nuclei of HeLa cells infected with adenovirus 5 (Ad5) carries out the semiconservative replication of exogenously added Ad5 DNA in vitro. Maximal DNA synthesis is observed when DNA-protein complex, isolated from Ad5 virions, is added as template. DNA-protein complex from virions of the closely related virus, adenovirus 2, is also active. In contrast, very little in vitro DNA synthesis is observed when deproteinized Ad5 DNA or DNA from a heterologous source (bacteriophage T7) is added as template. The product of the in vitro reaction consists of long Ad5 DNA strands that are hydrogen-bonded, but not covalently linked, to the input DNA template. During the course of the in vitro reaction, branched molecules with structural features identical to in vivo replication intermediates are formed. These findings support the conclusion that replication in the in vitro system closely resembles adenovirus DNA replication in vivo. The system provides an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.


Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S. We have previously found that the inhibitory effect of hemoglobin F (Hb F) on the polymerization of Hb S proceeds via the formation of asymmetrical hybrid tetramers of the type alpha2betasgamma. Examination of the gelling properties of binary mixtures of Hb S and several Hb variants now shows that, among the gamma chain amino acid residues that differ from those of the beta chain, residues gamma80 (EF4) and gamma87 (F3) are at least partly responsible for this inhibition. Furthermore, we find that mixing Hb A2(alpha2delta2) with Hb S strongly inhibits gelling to an extent similar to that seen with Hb S/Hb F mixtures; this inhibition is attributable to amino acid differences between the delta and beta chain sequences at positions delta22 (B4) and delta87 (F3). Therefore, residues 22, 80, and 87 of the beta chain appear to be involved in intermolecular contact sites that stabilize the deoxy Hb S polymers.


Temperature-sensitive mutation in the initiation codon of the rIIB gene of bacteriophage T4. We have determined the sequence of a ribosome-protected region of T4 rIIB mRNA labeled in vivo. The rIIB mutant HD263, which is temperature sensitive for translation, has an altered initiation codon (AUA instead of AUG). Because wild-type and HD263 proteins have the same fMet peptide, this AUA is used to initiate translation in the mutant rIIB mRNA. We have also identified a six-base sequence which is complementary to the 3" end of the 16S rRNA.


Degradation of abnormal proteins in intact mouse reticulocytes: accumulation of intermediates in the presence of bestatin. Incubation of intact mouse reticulocytes with bestatin (a competitive inhibitor of aminopeptidases) produced the accumulation of low molecular weight intermediates in the degradation of puromycinyl-peptides or analog-containing proteins that had been pulse labeled with L-[1-14C]leucine. A large fraction of the radioactive protein was degraded to trichloroacetic acid-soluble products within 10 min. In the presence of bestatin (0.5 mg/ml), one-fourth of these products appeared to be dipeptides, tripeptides, or both: they were resistant to ninhydrin at acid pH (a treatment that decarboxylates only free amino acids) except after intensive acid hydrolysis, and they eluted from a Sephadex G-10 column with an apparent average size of 300 daltons. These radioactive products did not appear if incorporation of the tracer was prevented by prior treatment with cycloheximide, demonstrating that they originated from polypeptide precursors. Thus, a peptidase inhibitor has been successfully used in the production of low molecular weight intermediates in the in vivo degradation of cellular proteins.


Labeling of complex III, with [35S]diazobenzenesulfonate: orientation of this electron transfer segment in the mitochondrial inner membrane. [34S]Diazobenzenesulfonate has been used to tag the surface-exposed polypeptides of isolated complex III. All nine different component polypeptides were labeled, indicating that each is at least partially exposed on the surface of the isolated, detergent-dispersed complex. Labeling studies were also conducted on the membrane-bound complex. Preparations of intact mitochondria and submitochondrial particles were separately labeled with [35S]diazobenzenesulfonate in order to determine the distribution of the polypeptides of complex III between the outer (cytoplasmic) and inner (matrix) surfaces of the mitochondrial inner membrane, respectively. Polypeptides II and III were the only components labeled in a significant amount in submitochondrial particles (i.e., from the matrix side). Polypeptides III, IV, and VI were heavily labeled in mitochondria (i.e., from the cytoplasmic side). Polypeptides I,II, V, and VII were also labeled in mitochondria but to a much lesser extent. Polypeptides VIII and IX were not significantly labeled from either side of the membrane. The labeling data and information obtained from previous crosslinking studies [Smith, R.J. & Capaldi, R.A. (1977) Biochemistry 16, 2629-2633] are used to derive a picture of the arrangement of complex III in the mitochondrial inner membrane.


Self-catalyzed destruction of cytochrome P-450: covalent binding of ethynyl sterols to prosthetic heme. The hepatic pigment accumulated as a consequence of the self-catalyzed destruction of cytochrome P-450 by norethisterone (17-hydroxy-19-nor-17 alpha-pregn-4-en-20-yn-3-one), after acidic methylation and purification, consists of two virtually identical, probably isomeric, porphyrins. Radiolabeled norethisterone is incorporated into both porphyrin products. The major of the two porphyrins exhibits a mass spectrometric molecular ion exactly equivalent to the sum of norethisterone and dimethylprotoporphyrin IX, less two hydrogen atoms: unequivocably demonstrating covalent association of the sterol with this porphyrin in a 1:1 ratio. Cytochrome P-450 is therefore destroyed by self-catalyzed addition of norethisterone to its heme prosthetic group. Cytochrome P-450 is also destroyed by norgestrel (13-ethyl-17-hydroxyl-18, 19-dinor-17 alpha-pregn-4-en-20-yn-3-one) and by 1-ethynylcyclohexanol but not by 17-hydroxy-19-nor-17alpha-pregn-4,20-dien-3-one. The destructive potential is thus clearly a property of the propargylic alcohol function. A mechanism involving enzymatic oxidation of the triple bond is postulated.


Heterogeneous nuclear RNA double-stranded regions probed in living HeLa cells by crosslinking with the psoralen derivative aminomethyltrioxsalen. The psoralen derivative aminomethyltrioxsalen (AMT, 4'-aminomethyl-4,5',8-trimethylpsoralen) has been employed as a probe for heterogeneous nuclear RNA (hnRNA) double-stranded regions in experiments with living HeLa cells. hnRNA ribonucleoprotein (hnRNP) particles were purified from untreated or AMT-treated cells after irradiation with 365-nm light, and double-stranded hnRNA regions (dsRNA) were isolated by RNase A + T1 digestion of hnRNP, followed by preparative Cs2SO4 isopycnic centrifugation. The purified, hnRNP-derived dsRNA was then assayed for interstrand crosslinks by measurement of its "snapback" to RNase-resistant form after thermal denaturation. By this procedure, the amount of crosslinked dsRNA was found to be increased 3- to 7-fold in cells exposed to AMT in vivo. The levels of crosslinking in vivo compared favorably with those observed in model experiments with pure dsRNA in vitro. These results establish that double-stranded hnRNA regions exist in the living cell, and they further demonstrate that these base-paired regions are organized as rather accessible sites within the nucleus.


Stability of lipid vesicles in tissues of the mouse: a gamma-ray perturbed angular correlation study. The rate of phospholipid vesicle disruption in specific tissues of the mouse was followed by gamma-ray perturbed angular correlation (PAC) spectroscopy. In these studies, high levels of 111In-nitrilotriacetic acid complex are contained in unilamellar vesicles consisting of distearoyl phosphatidylcholine, cholesterol, and small amounts of other lipids which modify the surface properties. The PAC technique monitors the extent of vesicle breakup by measuring a time-integrated perturbation factor, less than G22 (infinity) greater than. As the vesicles are broken open in vivo, the released 111In3+ ions quickly bind to macromolecules and the less than G22 (infinity) greater than value decreases substantially. After administration of vesicles by various routes (intravenous, intraperitoneal, subcutaneous, and oral), the radioactivity and less than G22 (infinity) greater than values were determined for several tissues at intervals up to 24 hr. We conclude from these data that (i) the PAC technique in conjunction with standard gamma counting methods provides unique information on the condition and location of vesicles in specific tissues, (ii) significant differences in vesicle integrity are found in various tissues, and (iii) both the means of administration and the presence of surface charge affect the vesicle stability and distribution. The carbohydrate analogues of cholesterol affect vesicle stability but not distribution.


Collagen reduces glycosaminoglycan degradation by cultured mammary epithelial cells: possible mechanism for basal lamina formation. Collagenous substrates are reported to promote the accumulation of extracellular matrix materials by epithelia in culture. Glycosaminoglycan (GAG) metabolism is compared in secondary cultures of mouse mammary epithelial cells maintained on plastic or type I collagen gel substrates. The incorporation of 35SO42- into GAG during brief labeling indicates no difference between substrates in the rate of GAG synthesis. During prolonged labeling, however, accumulation of [35S]GAG in cultures on colllagen exceeds that of cultures on plastic. This increased accumulation is due to a markedly reduced rate of GAG degradation. GAG degradation does not occur in the medium, indicating that degradation is localized to the cells. The cultures on collagen contain a slowly degrading cell-associated [35S]GAG pool and a ruthenium red-stained basal lamina, neither of which is present in cultures on plastic. The cell-associated [35S]GAG in cultures on collagen is, in part, localized to the site of the ultrastructurally identified basal lamina. Formation of the basal lamina, therefore, may result from collagen-mediated reduction in the degradation of GAG-containing molecules.


Change in nuclear potassium electrochemical activity and puffing of potassium-sensitive salivary chromosome regions during Chironomus development. Changes in nuclear K+ electrochemical activity and total nuclear K+ content in salivary glands of Chironomus tentans were measured with ion-selective microelectrodes based on valinomycin and with flameless atomic absorption spectrometry, respectively. The K+ activity increased by a factor of 2.6 and the total K+, by a factor of 1.5 as oligopausing larvae developed into prepupae. The extent of decondensation (puffing) of K+-sensitive regions in the polytene chromosomes underwent a parallel increase during this developmental event. In vitro culture of glands from oligopausing larvae resulted in similar changes with respect to nuclear K+ activity and puffing.


Desensitization of the insulin receptor at an early postreceptor step by prolonged exposure to antireceptor antibody. We have used an adipocyte-like cell line, the 3T3-L1 fatty fibroblasts, to compare acute and chronic effects of autoantibodies directed against the insulin receptor. Acute exposure of the cells in tissue culture to the antibodies resulted in a blockade of insulin binding and stimulation of 2-deoxy-glucose transport and glucose oxidation. Maximal acute effects were reached within 30-120 min. Subsequently, the stimulatory response decayed and, after 6 hr in the continuous presence of the antibodies, basal glucose metabolism had returned to the level observed with unexposed cells and a state of severe insulin resistance prevailed. In contrast to the decay of bioresponse, no change in insulin binding was detectable over the same time period. The mechanism of desensitization seemed to involve events early after insulin binding to receptor because cells exposed to antibody for prolonged periods of time, although unresponsive to insulin and antireceptor antibodies, responded normally to both spermine and vitamin K(5), agents that stimulate glucose metabolism independently of the insulin receptor. These data suggest that prolonged or continuous occupancy of the insulin receptor by a ligand, in this case antireceptor antibodies, does not produce a continuous biological response. Instead, there is desensitization at some early step in the pathway for insulin action. These observations have important implications with respect to the mechanism of insulin action and to other situations in which there is long-term exposure of cells to antibodies that react with membrane components.


Decreased fidelity of DNA polymerase activity during N-2-fluorenylacetamide hepatocarcinogenesis. alpha and beta DNA polymerases (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) were isolated from nuclear and cytoplasmic fractions of rat livers exposed to a carcinogenic regimen with the hepatocarcinogen N-2-fluorenylacetamide and from 24-hr regenerating liver. The fidelity of polymerization of these enzymes was compared by determining the incorporation of noncomplementary deoxyribonucleoside triphosphates (misincorporation) on a poly(dA-dT).poly(dA-dT) template, with MnCl2 and MgCl2 as divalent cations. Our initial studies indicate that the cytoplasmic alpha polymerases from carcinogen-exposed rat livers were strikingly error-prone whereas the nuclear and cytoplasmic beta polymerases retained their fidelity throughout the feeding cycles. The misincorporation was significantly accentuated by MnCl2 compared with that obtained with MgCl2 as divalent cation. The products were sensitive to pancreatic DNase I digestion, indicating that the noncomplementary bases had been incorporated by the polymerization process. Nuclear alpha polymerase showed some degree of infidelity but less than that of cytoplasmic alpha polymerase.


Phosphorylation of subunit proteins of intermediate filaments from chicken muscle and nonmuscle cells. The phosphorylation of the subunit proteins of intermediate (10-nm) filaments has been investigated in chicken muscle and nonmuscle cells by using a two-dimensional gel electrophoresis system. Desmin, the 50,000-dalton subunit protein of the intermediate filaments of muscle, had previously been shown to exist as two major isoelectric variants-alpha and beta-in smooth, skeletal, and cardiac chicken muscle. Incubation of skeletal and smooth muscle tissue with (32)PO(4) (3-) reveals that the acidic variant, alpha-desmin, and three other desmin variants are phosphorylated in vivo and in vitro. Under the same conditions, minor components of alpha- and beta-tropomyosin from skeletal muscle, but not smooth muscle, are also phosphorylated. Both the phosphorylated desmin variants and the nonphosphorylated beta-desmin variant remain insoluble under conditions that solubilize actin and myosin filaments, but leave Z-discs and intermediate filaments insoluble. Primary cultures of embryonic chicken muscle labeled with (32)PO(4) (3-) possess, in addition to the desmin variants described above, a major nonphosphorylated and multiple phosphorylated variants of the 52,000-dalton, fibroblast-type intermediate filament protein (IFP). Filamentous cytoskeletons, prepared from primary myogenic cultures by Triton X-100 extraction, contain actin and all of the phosphorylated and nonphosphorylated variants of both desmin and the IFP. Similarly, these proteins are the major components of the caps of aggregated 10-nm filaments isolated from the same cell cultures previously exposed to Colcemid. These results demonstrate that a nonphosphorylated and several phosphorylated variants of desmin and IFP are present in assembled structures in muscle and nonmuscle cells.


Comparison of primary and secondary stimulation of male rats by estradiol in terms of prolactin synthesis and mRNA accumulation in the pituitary. Male rats received acute or chronic primary or acute secondary stimulation with estradiol, and the effects on pituitary prolactin synthesis and its mRNA accumulation were examined. Prolactin synthesis was determined by the in vitro incorporation of [(3)H]leucine into prolactin over a period of 1 hr. Prolactin mRNA was measured both by cell-free translation in a nuclease-treated rabbit reticulocyte lysate and by hybridization to the complementary DNA. The latter two methods gave similar results under all experimental conditions. Acute primary stimulation with estradiol produced a significant increase in pituitary prolactin mRNA accumulation at 12 hr, which further increased by 2- to 3-fold over the next 48 hr. In contrast, no increase in prolactin synthesis was observed during the first 24 hr. Chronic stimulation with estradiol induced increases of both prolactin synthesis and prolactin mRNA that were quantitatively indistinguishable over the period of 1-4 weeks, reaching a plateau at 5-fold the basal values. By the 13th day after withdrawal of therapy both prolactin synthesis and mRNA had returned to the prestimulation levels. When the effects of estradiol on previously unexposed and estrogen withdrawn animals were compared, it was found that secondary stimulation not only produced a more rapid accumulation of the prolactin mRNA but also abolished the lag period of prolactin synthesis observed during the primary estrogen stimulation. These data demonstrate a lag in the endogenous translation of newly accumulated pituitary prolactin mRNA translatable in vitro after primary estrogen stimulation of male rats. The mechanism for the abolition of this lag during the secondary stimulation is now known.


Sodium ascorbate potentiates the growth inhibitory effect of certain agents on neuroblastoma cells in culture. Mouse neuroblastoma (NB) cells in culture were more sensitive to sodium L-ascorbate than were rat glioma cells by the criterion of growth inhibition (due to cell death and reduction in cell division). Sodium L-ascorbate at nonlethal concentrations potentiated the effect of 5-fluorouracil (FUra), x-irradiation, bleomycin, RO20-1724, prostaglandin E1, and sodium butyrate on NB cells but did not produce such an effect on glioma cells. Sodium L-ascorbate did not enhance the effect of vincristine, 6-thioguanine, or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) except at higher drug doses and it reduced the cytotoxic effect of methotrexate and 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) on NB cells. Sodium D-ascorbate produced effects similar to those produced by sodium L-ascorbate on NB cells. L-Ascorbic acid-2-sulfate (barium salt) affected neither the growth rate nor the effect of 5-FUra on NB cells. Glutathione, a reducing agent, was more toxic to NB cells in comparison to D- OR L-ascorbate; however, at a similar concentration it failed to potentiate the effect of 5-FUra on NB cells.


Heme biosynthesis in Friend erythroleukemia cells: control by ferrochelatase. The activities of the enzymes of heme biosynthesis (except protoporphyrin oxidase) have been followed during the induction of Friend cells in culture. All the enzyme activities increased after induction with dimethyl sulfoxide. The activities of the intermediate enzymes were much higher than those of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37], the initial enzyme, or ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the final enzyme of the pathway. Ferrochelatase activity was not detectable in the uninduced cell. delta-Aminolevulinate synthase activity increased during the first 24 hr of induction; porphobilinogen deaminase activity began to increase after 48 hr and ferrochelatase activity, after 72 hr. However, the induction of heme synthesis followed the same time course as that of ferrochelatase activity, not that of delta-aminolevulinate synthase activity. The cellular growth medium was found to contain traces of protoporphyrins. Thus, ferrochelatase is shown to be rate limiting for heme synthesis during early stages of Friend cell induction. A Friend cell variant (Fw), which is not inducible except in the presence of exogenous hemin, was also studied. All the enzymes of heme synthesis except ferrochelatase were inducible by butyric acid. Ferrochelatase was not inducible by butyric acid or hemin plus butyric acid. These cells also excrete protoporphyrin, The failure to induce ferrochelatase activity is believed to be the cause of, not a consequence of, the noninducibility of this cell line.


Electrical currents through full-grown and maturing Xenopus oocytes. An extracellular vibrating electrode was used to map the current pattern around Xenopus laevis oocytes. Current was found to enter the animal hemisphere and leave the vegetal hemisphere; in fully grown oocytes from which the follicle cells had been removed, the maximal current density was about 1 microamperemeter/cm2. This current decreased to nearly zero in response to progesterone and several other maturation-producing agents. In the case of progesterone, the decline began within a few minutes of the addition of the hormone and proceeded with a half-time of about 20 min. An analysis of the effects on the current of the removal or addition of various ions and drugs led to the inference that the major current-carrying ion was chloride and that the chloride permeability was controlled by calcium.


Molecular evolution of biomembranes: structural equivalents and phylogenetic precursors of sterols. Derivatives of one triterpene family, the hopane family, are widely distributed in prokaryotes; they may be localized in membranes, playing there the same role as sterols play in eukaryotes, as a result of their similar size, rigidity, and amphiphilic character. Their biosynthesis embodies many primitive features compared to that of sterols and could have evolved toward the latter once aerobic conditions had been established. Membrane reinforcement appears to be achieved in other prokaryotes by other mechanisms, involving either approximately 40-A-long rigid hydrocarbon chains terminated by one polar group acting like a peg through the double-layer or similar chains terminated by two polar groups acting like tie-bars across the membrane. These inserts can be tetraterpenes (e.g., carotenoids). The biophysical function of membrane optimizers appears to have evolved toward sterols by changes limited to only a few enzymatic steps of the same fundamental biosynthetic processes.


Genetic heterogeneity of "normal" human erythrocyte glucose-6-phosphate dehydrogenase: an isoelectrophoretic polymorphism. Quantitative determination of glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) activity was carried out in 214 male Nigerian children of 84 mothers with known Gd genotype. The relative intrasibship difference in G6PD activity (normalized to the lowest value within the sibship) was below 0.18 in all cases but one when the children were known to have the same Gd+ allele (identical by descent); whereas it was higher than 0.18 in 18 out of 33 sibships in which children might have had either of the two maternal (electrophoretically identical) Gd+ alleles. G6PD from 10 (8 G6PD B and 2 G6PD A) children belonging to four of the sibships possessing high quantitative variation in G6PD activity was partially purified and extensively characterized. The 8 G6PD type B samples fell unambiguously into two classes on the basis of Km values for glucose 6-phosphate (determined at variuos pH values), and KCl gradient elution from DEAE-Sephadex columns. The two types of G6PD B were resolved from an artificial mixture on a DEAE-Sephacel column. The two G6PD type A samples were also different from each other by the same criteria. We conclude that "normal" G6PD is genetically heterogeneous and that the structural Gd alleles concerned are all polymorphic in the Nigerian population. In this instance, a human enzyme polymorphism, not associated with enzyme deficiency, is revealed by an approach other than electrophoresis.


Resolution of products of the duplicated hemoglobin alpha-chain loci by isoelectric focusing. Using a high-resolution isoelectric focusing system, we have been able to separate the products of the individual "nonallelic+ mouse hemoglobin alpha-chain genes. The hemoglobins of mice of inbred strains that make two structurally different, but electrophoretically identical, alpha chains resolve into two bands on isoelectric focusing. The hemoglobin from other strains that make only one type of alpha chain forms a single band. Two new "alleles" of haplotypes of Hba (the alpha-chain complex locus) are described that code for hemoglobins with solubilities similar to that of the hemoglobin of strain C57BL/6J, but that give demonstrably different isoelectric focusing patterns. Because this method allows Hba typing of strains with the Hbbd or "diffuse" allele at the hemoglobin beta-chain locus, we have also been able to classify a number of previously untyped stocks, including some recently derived from feral populations. Among these mice we have found new representatives of most of the extant Hba genotypes. The demonstration that it is possible to detect genetic differences involving only neutral amino acid substitutions between proteins suggest that careful application of appropriate isoelectric focusing systems to the analysis of other proteins will greatly increase the sensitivity of our ability to detect genetic variation.


Xenogeneic gene expression in chimeric mice derived from rat--mouse hybrid cells. Thymidine kinase-deficient OTT6050 mouse teratocarcinoma cells were fused with hypoxanthine phosphoribosyltransferase-deficient Fu5AH rat hepatoma cells by means of inactivated Sendai virus. The resulting hybrid cells, which were selected in hypoxanthine/aminopterin/thymidine medium, retained almost all of the mouse chromosomes and various numbers of rat chromosomes, and showed many chromosomal rearrangements. The hybrid cells, as well as both parental lines, formed tumors after subcutaneous injection into athymic nude mice. Single rat--mouse hybrid cells from a clonally established subline were transplanted into C57BL6/J mouse blastocysts carrying many genetic markers suitable for the detection of hybrid cell-derived tissue contributions. From 144 blastocysts, each of which was injected with a hybrid cell and then surgically transferred to the uterus of a pseudopregnant foster mother, 62 adult mice developed without any visible coat mosaicism. However, three of these mice showed internal hybrid-cell participation in their livers and a limited number of organs of endomesodermal origin. A tumor classifiable as hemangio endothelioma was found in the liver, the only mosaic tissue, of one of the chimeric mice. Nine different rat-specific enzyme variants were detected in the mosaic organs. A considerable number of variations concerning the presence and quantitative activity of the foreign gene products probably resulted from chromosomal segregation, tissue-specific gene activity, or dosage compensation during differentiation in vivo. Our results demonstrate that cultured malignant rat--mouse hybrid cells differentiate normally and become functionally integrated during development. The appearacne in vivo of certain rat-specific gene products that are not found in the hybrid cells under conditions in vitro indicates differential gene expression of the introduced xenogeneic chromosomes.


Regulation of macrophage migration by products of the complement system. Agents formerly shown to induce rapid macrophage spreading were examined for their ability to modify the migration of macrophages in the capillary tube assay. Products of the activation of the contact phase of blood coagulation as well as the purified component Bb, the large cleavage fragment of factor B of the alternative complement pathway produced a dose-dependent inhibition of migration. In addition, inflammatory macrophages elicited with either a lipopolysaccharide endotoxin or thioglycollate medium exhibited rapid spreading and inhibited migration, whereas resident cells did not. A close correlation existed, therefore, between enhanced spreading and inhibited migration under both in vitro induced and in vivo situations. Cleavage products of component C5 of the classical complement pathway enhanced macrophage migration and did not alter spreading. In mixtures of C5 cleavage products and Bb, the predominant peptide determined the outcome of the reaction. Factor B, a normal secretory product of macrophages, may represent a common substrate for several of the proteases that induce spreading, inhibit migration, and lead to the generation of the enzymatically active fragment Bb.


Nucleolar antigen found in several human tumors but not in the nontumor tissues studied. Rabbit antibodies to nucleoli isolated from HeLa cells produced bright nucleolar fluorescence in HeLa cells by the indirect immunofluorescence technique. After absorption with fetal bovine serum and placental nuclei, the IgG still produced bright nucleolar fluorescence in 12 human tumor cells including HeLa, HEp-2, cultures of prostate and mammary carcinomas, the Goldenberg GW-39 colon tumor, and biopsy specimens of prostatic, adrenal cortical, thyroid, and squamous cell carcinomas, a hairy cell leukemia of the spleen, a hepatic metastasis of an adenocarcinoma of the colon, and an osteogenic sarcoma. Bright nucleolar fluorescence was not produced in nine nontumor human cells including biopsy specimens of bone marrow, kidney, placenta, thyroid, liver, and prostate, peripheral blood buffy coat, and cultures of normal skin fibroblasts. Nucleolar fluorescence with the absorbed IgG was prevented in HeLa cells by pretreatment of the cells with acid, base, and proteases but not by pretreatment with nucleases; absorption of this IgG with extracts of HeLa nucleoli prevented the nucleolor fluorescence in HeLa and other human tumor cells.


Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins. The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid--protein complexes were formed with the MAC precursor components C5b--6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b--7, C5b--8, C5b--9, and the MAC (C5b--9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b--7, 399:1, C5b--5, 841:1; C5b--9, 918:1; and C5b--9 dimer, 1460:1. Electron microscopy of the isolated phospholipid--protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid--protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.


Insertion of Ia and H-2 alloantigens into model membranes. The study of immune phenomena dependent on the major histocompatibility complex (MHC) would be greatly simplified by the use of MHC antigen-containing liposomes in various functional systems. Towards this end, we have constructed unilamellar phosphatidylcholine liposomes containing H-2 and Ia antigens. These molecules were not simply trapped within the aqueous compartment of the liposome as assessed by their accessibility to papain digestion. They were shown to be integrally inserted in the liposome bilayer because they could not be dissociated from the liposome with high salt and EDTA concentrations but could be solubilized by detergent. A sensitive radioimmunoassay showed that the Ia molecules were antigenically active in the liposome environment. Both Ia and H-2 antigens could be immunoprecipitated from detergent-solubilized liposomes. By comparing liposome-associated Ia activity in the presence and absence of detergent and by showing accessibility of the Ia antigens to papain, it was concluded that the majority of Ia antigens were exposed on the external surface of the liposome. These results suggest that the orientation of MHC antigens in liposomes closely parallels their natural orientation in the cell membrane.


IgM receptors on human lymphocytes: detection by direct binding. Radioiodinated IgM isolated from a patient with Waldenström macroglobulinemia binds with high avidity (apparent Ka = 2.5 x 10(9) M-1) to freshly isolated human peripheral blood lymphocytes. Binding occurred through the Fc region of the molecule and involved a receptor specific for immunoglobulins of the IgM class. The avidity and specificity of this binding are consistent with a biologic role for IgM receptors on lymphocytes.


Chemical carcinogens produce mutations to ouabain resistance in transformable C3H/10T1/2 Cl 8 mouse fibroblasts. Chemical carcinogens induce mutations to ouabain resistance in the transformable mouse fibroblast cell line C3H/10T1/2 Cl 8. The mutant phenotype is stable and heritable in the absence of selective agent, and dose--response curves for mutant frequency were obtained with N-menthyl-N'-nitro-N-nitrosoguanidine, 3-methylcholanthrene, benzo[a]-pyrene, N-acetoxy-N-2-acetylaminofluorene, and the anti-7,8-diol-9,10-epoxide of benzo[a]pyrene. The ratio of the malignant transformation frequency to the mutation frequency was 12 for benzo[a]pyrene and 21 for N-acetoxy-N-2-acetylaminofluorene. The development of the mutational assay reported here allows the use of this permanent cell line for comparison of mutation and transfomration frequencies and as a screening system for xenobiotics that pose mutagenic or carcinogenic hazards to mammalian cells.


Comparison of structure and function of human erythrocyte and human muscle actin. Human erythrocyte actin and human skeletal muscle actin were purified by acetone powder extraction and gel filtration. Pure human erythrocyte actin resembles muscle actin in its polymerization and depolymerization by phalloidin, cytochalasin B, and DNase I, in its peptide mapping pattern, and in the amino acid composition of corresponding peptides. Isoelectric focusing gel analysis showed that human erythrocyte actin exists in the beta/gamma form, but muscle actin is in the alpha form. Abnormal deformability of resealed erythrocyte membranes was observed after incorporation of the actin-specific agents, phalloidin and DNase I, suggesting that erythrocyte actin might function as a membrane structural element to maintain erythrocyte membrane deformability.


Quantitative studies of ferritinlike iron in erythrocytes of thalassemia, sickle-cell anemia, and hemoglobin Hammersmith with Mössbauer spectroscopy. By using the technique of recoil-free absorption (Mössbauer effect) in iron, we found large amounts of iron, yielding a well-defined spectrum different from that of oxy- or deoxyhemoglobin, in whole erythrocytes of 13 patients with beta-thalassemia major and intermedia, 3 with hemoglobin H disease, 2 with sickle-cell anemia, and 1 with unstable hemoglobin Hammersmith. The Mössbauer spectra at various temperatures of this additional component of iron were found to be identical to spectra obtained from isolated ferritin or hemosiderin. This observation, together with additional arguments, strongly suggests that the compound responsible for the additional subspectrum is an iron storage protein, ferritin or hemosiderin. The amounts of ferritinlike iron were comparable to those of hemoglobin iron and were particularly large in reticulocytes. No ferritinlike iron was detected in patients with severe autoimmune hemolytic anemia and pernicious anemia. The large quantities of ferritinlike iron in hemoglobinopathies are probably due to intracellular hemoglobin denaturation and the consequent release of excess iron.


Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent. To investigate the earliest steps of the intrinsic clotting pathway, Hageman factor (Factor XII) was exposed to Sephadex gels to which ellagic acid had been adsorbed; Hageman factor was then separated from the gels and studied in the fluid phase. Sephadex-ellagic acid-exposed Hageman factor, whether purified or in plasma, activated plasma thromboplastin antecedent, but only when high molecular weight kininogen was presnet. In the absence of plasma prekallikrein, maximal activation of plasma thromboplastin antecedent was slightly delayed in plasma, a delay not observed with similarly treated purified Hageman factor. Thus, high molecular weight kininogen was needed for expression of Hageman factor's clot-promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor and plasma thromboplastin antecedent.


Transformation of DBA/2 mouse fetal liver cells infected in vitro by the anemic strain of Friend leukemia virus. Fetal liver cells of DBA/2 mice were infected with the anemic strain of Friend leukemia virus (FLV-A), which has no spleen focus-forming virus (SFFV) activity. The infected cells were grown in medium with or without erythropoietin. Transformed lines were isolated only from the infected cultures that had been treated with erythropoietin at the time of their initiation. The properties of three permanent cell lines in serial passage for over 2 years are described. Each has an aneuploid karyotype. Only the immature hematopoietic cells of the first line have metacentric chromosomes. They grow in suspension, as do the erythroleukemic lines derived from leukemic spleens of FLV-infected mice, and clone on agar. They produce tumors resembling reticulum cell sarcomas upon subcutaneous inoculation into syngeneic hosts. Stimulation of differentiation induced after treatment with dimethyl sulfoxide identifies the cells of the first line as being erythroid in origin. The two other lines are adherent and epithelioid in appearance. These lines may have originated from the nonhematopoietic cells present in fetal liver. No tumors were produced after the subcutaneous inoculation of 10(6) cells. All three lines synthesize virus. The virus is attenuated for leukemogenicity and has no SFFV activity. The transforming event appears to be specific, because fetal liver cells from C57BL/6 mice, which are resistant to the induction of leukemia by FLV, were not affected by the virus. Malignant transformation of erythroid cells by FLV-A in vitro confirms the in vivo findings that SFFV may not be a necessary prerequisite for the induction of erythroleukemia in susceptible hosts.


Identification of inosine and hypoxanthine as endogenous ligands for the brain benzodiazepine-binding sites. Two endogenous ligands for the brain benzodiazepine-binding sites were isolated from bovine brain through gel filtration, paper electrophoresis, and paper chromatography. These ligands were identified as inosine and hypoxanthine, and both had a higher affinity for the brain benzodiazepine-binding sites than for benzodiazepine sites in some peripheral tissues. They did not bind to any other receptors tested, such as the opiate, muscarinic cholinergic, gamma-aminobutyric acid, and beta-adrenergic receptors. Both inosine and hypoxanthine competitively inhibited the binding of [3H]diazepam to the brain binding site.