Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 1976"
TL;DR: A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma and should be a useful model system for neurobiological and neurochemical studies.
Abstract: A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.
5,409Â citations
TL;DR: Viroids are uncoated infectious RNA molecules pathogenic to certain higher plants and exhibit high thermal stability, cooperativity, and self-complementarity resulting in a rod-like native structure.
Abstract: Viroids are uncoated infectious RNA molecules pathogenic to certain higher plants. Four different highly purified viroids were studied. By ultracentrifugation, thermal denaturation, electron microscopy, and end group analysis the following features were established: (i) the molecular weight of cucumber pale fruit viroid from tomato is 110,000, of citrus exocortis viroid from Gynura 119,000, of citrus exocortis viroid from tomato 119,000 and of potato spindle tuber viroid from tomato 127,000. (ii) Viroids are single-stranded molecules. (iii) Virods exhibit high thermal stability, cooperativity, and self-complementarity resulting in a rod-like native structure. (iv) Viroids are covalently closed circular RNA molecules.
1,565Â citations
TL;DR: It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament.
Abstract: Chromatin prepared by brief digestion of nuclei with micrococcal nuclease, and extracted in 0.2 mM EDTA, appears in the electron microscope as filaments of about 100 A diameter which coil loosely. In 0.2 mM Mg++ these "nucleofilaments" condense into a supercoil or solenoidal structure of pitch about 110 A corresponding to the diameter of a nucleofilament. It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament. The solenoidal structure appears to need histone H1 for its stabilization. Under certain conditions, isolated nucleosomes can also aggregate into a similar structure. The solenoidal structure can be correlated with the "thread" of diameter about 300 A observed by other workers in nuclei.
1,126Â citations
TL;DR: Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase by purified from Escherichia coli cells, and the final superhelix density of the DNA can be considerably greater than that found in intracellularly super coiled DNA.
Abstract: Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase. This enzyme has been purified from Escherichia coli cells. The reaction requires ATP and Mg++ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin E1, phage lambda, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA.
1,108Â citations
TL;DR: Purified rat liver peroxisomes contain a cyanide-insensitive fatty acyl-CoA oxidizing system that uses O2 and NAD as electron acceptors, and the activity of this system is increased approximately one order of magnitude in rats treated with clofibrate.
Abstract: Purified rat liver peroxisomes contain a cyanide-insensitive fatty acyl-CoA oxidizing system that uses O2 and NAD as electron acceptors. The system was detected by the ability of added palmitoyl-CoA to elicit O2 consumption, H2O2 production, and O2-dependent NAD reduction. The activity of this system is increased approximately one order of magnitude in rats treated with clofibrate, a hypolipidemic drug known to cause peroxisomal proliferation.
1,027Â citations
TL;DR: It is concluded that a single hydrogen bond is inadequate for uniquely identifying any particular base pair, as this leads to numerous degeneracies, but using two hydrogen bonds, fidelity of base pair recognition may be achieved.
Abstract: The base pairs in double helical nucleic acids have been compared to see how they can be recognized by proteins. We conclude that a single hydrogen bond is inadequate for uniquely identifying any particular base pair, as this leads to numerous degeneracies. However, using two hydrogen bonds, fidelity of base pair recognition may be achieved. We propose specific amino-acid side chain interactions involving two hydrogen bonds as a component of the recognition system for base pairs. In the major groove we suggest that asparagine or glutamine binds to adenine of the base pair or arginine binds to guanine. In the minor groove, we suggest an interaction between asparagine or glutamine with guanine of the base pair. We also discuss the role that ions and other amino-acid side chains may play in recognition interactions.
1,015Â citations
TL;DR: Although the cationized LDL entered the cell through a mechanism independent of the LDL receptor, the cholesterol liberated from the degradation of the lipoprotein became available for suppression of cholesterol synthesis and stimulation of cholesteryl ester formation in the homozygous familial hypercholesterolemia fibroblasts.
Abstract: Cultured fibroblasts derived from patients with homozygous familial hypercholesterolemia, which lack functional low density lipoprotein (LDL) receptors, fail to bind, take up, or degrade the lipoprotein with high affinity; therefore LDL-cholesterol is not made available for suppression of cholesterol synthesis or activation of cholesteryl ester formation. When LDL was given a positive charge by reaction with N,N-dimethyl-1,3-propanediamine (cationized LDL), the rate of degradation of the lipoprotein was increased by more than 100-fold in the homozygous familial hypercholesterolemia fibroblasts. Degradation of cationized LDL was inhibited by chloroquine, suggesting that it occurred in cellular lysosomes. Although the cationized LDL entered the cell through a mechanism independent of the LDL receptor, the cholesterol liberated from the degradation of the lipoprotein became available for suppression of cholesterol synthesis and stimulation of cholesteryl ester formation in the homozygous familial hypercholesterolemia fibroblasts. The rate of degradation of albumin by fibroblasts was also increased by more than 100-fold when this protein was coupled to N,N-dimethyl-1,3-propanediamine. The ability to deliver a protein to lysosomes by giving it a strong positive charge may have potential relevance not only to familial hypercholesterolemia, but also to inborn errors of metabolism that involve deficiencies in lysosomal enzymes.
988Â citations
TL;DR: Comparison of the observed transition curves with theoretical curves calculated from the calorimetrically determined phase diagrams supports a literal interpretation of the phase diagrams.
Abstract: High sensitivity differential scanning calorimetry is applied to the study of the thermotropic behavior of mixtures of synthetic phospholipids in multilamellar aqueous suspensions. The systems dimyristoylphosphatidylcholine dipalmitoylphosphatidylcholine, and dimyristoylphosphatidylethanolamine-distearoylphosphatidylcholine, although definitely nonideal, exhibit essentially complete miscibility in both gel and liquid crystalline states, while the system dilauroylphosphatidylcholine-distearoylphosphatidylcholine is monotectic with lateral phase separation in the gel state. Comparison of the observed transition curves with theoretical curves calculated from the calorimetrically determined phase diagrams supports a literal interpretation of the phase diagrams.
859Â citations
TL;DR: Evidence that chemical carcinogens and radiation, likely to initiate most human cancer and genetic defects do so by damage to DNA is discussed, suggesting a central role in a program of prevention.
Abstract: About 300 carcinogens and non-carcinogens of a wide variety of chemical types have been tested for mutagenicity in the simple Salmonella/microsome test. The test uses bacteria as sensitive indicators of DNA damage, and mammalian liver extracts for metabolic conversion of carcinogens to their active mutagenic forms. There is a high correlation between carcinogenicity and mutagenicity: 90% (157/175) of the carcinogens were mutagenic in the test, including almost all of the known human carcinogens that were tested. Despite the severe limitations inherent in defining non-carcinogenicity, few "non-carcinogens" showed any degree of mutagenicity [McCann et al. (1975) Proc. Nat. Acad. Sci. USA 72, 5135-5139]. In the present paper, carcinogens negative in the test andapparent false positives are discussed. We also discuss evidence that chemical carcinogens and radiation, likely to initiate most human cancer and genetic defects do so by damage to DNA. The Salmonella test can play a central role in a program of prevention: to identify mutagenic chemicals in the environment (all indications are there are many) and to aid in the development of non-mutagenic products to prevent future human exposure.
823Â citations
TL;DR: The identity of gamma-carboxyglutamic acid in the bovine protein was established by mass spectroscopy on the unknown amino acid isolated from alkaline hydrolysates.
Abstract: A gamma-carboxyglutamic acid-containing protein has been purified from the calcified tissues of several vertebrates. The presence of three-gamma-carboxyglutamic acid residues in the bovine protein was established by alkaline hydrolysis and amino acid analysis, a method based upon studies with synthetic gamma-carboxyglutamic acid. The identity of gamma-carboxyglutamic acid in the bovine protein was established by mass spectroscopy on the unknown amino acid isolated from alkaline hydrolysates.
761Â citations
TL;DR: The results were interpreted to mean that the Vk and Ck genes are joined to form a contiguous polynucleotide stretch during differentiation of lymphocytes, which occurs in both of the homologous chromosomes.
Abstract: A high-molecular-weight DNA from Balb/c mouse early embryo or from MOPC 321 plasmacytoma (a k-chain producer) was digested to completion with Bacillus amyloliquefaciens strain H restriction enzyme (BamH I). The resulting DNA fragments were fractionated according to size in preparative agarose gel electrophoresis. DNA fragments carrying gene sequences coding for the variable or constant region of k chains were detected by hybridization with purified, 125I-labeled, whole MOPC 321 K MRNA and with its 3'-end half. The pattern of hybridization was completely different in the genomes of embryo cells and of the plasmacytoma. The pattern of embryo DNA showed two components, one of which (molecular weight=6.0 million) hybridized with C-gene sequences and the other (molecular weight=3.9 million) with V-gene sequences. The pattern of the tumor DNA showed a single component that hybridized with both V-gene and C-gene sequences and that is smaller (molecular weight=2.4 million) than either of the components in embryo DNA. The results were interpreted to mean that the Vk and Ck genes, which are some distance away from each other in the embryo cells, are joined to form a contiguous polynucleotide stretch during differentiation of lymphocytes. Such joining occurs in both of the homologous chromosomes. Relevance of these findings with respect to models for V-C gene joining, activation of a specific V k gene, and allelic exclusion in immunoglobulin gene loci is discussed.
TL;DR: It is concluded that DNA gyrase controls the supercoiling of DNA in E. coli.
Abstract: Novobiocin and coumermycin are known to inhibit the replication of DNA iing of DNA catalyzed by E. coli DNA gyrase, a recently discovered enzyme that introduces negative superhelical turns into covalently circular DNA. The activity of DNA gyrase purified from a coumermycin-resistant mutant strain is resistant to both drugs. The inhibition by novobiocin of colicin E1 plasmid DNA replication in a cell-free system is partially relieved by adding resistant DNA gyrase. Both in the case of coliclls. DNA molecules which are converted to the covalently circular form in thepresence of coumermycin remain relaxed, instead of achieving their normal supercoiled conformation. We conclude that DNA gyrase controls the supercoiling of DNA in E. coli.
TL;DR: The amount of clathrin in lymphoma cells suggests that coated vesicles transfer substantial amounts of membrane within cells, not necessarily in association with a secretory process.
Abstract: Coated vesicles have been purified from brain, adrenal medulla, and a nonsecreting lymphoma cell line. A single major protein species, clathrin, with an apparent molecular weight of 180,000, forms the coat of all these vesicles. Peptide mapping suggests that the amino acid sequence of clathrin is conserved, irrespective of tissue or species studied. Coated vesicles of different sizes are found. The coats are constructed with variable numbers of clathrin subunits, arranged in closed networks of hexagons and pentagons. The amount of clathrin in lymphoma cells suggests that coated vesicles transfer substantial amounts of membrane within cells, not necessarily in association with a secretory process.
TL;DR: Analysis of the survival-time curves indicates that deaths occur for about 90% of the ascorbate-treated patients at one-third the rate for the controls and that the other 10% have a much greater survival time, averaging more than 20 times that for the Controls.
Abstract: Ascorbic acid metabolism is associated with a number of mechanisms known to be involved in host resistance to malignant disease. Cancer patients are significantly depleted of ascorbic acid, and in our opinion this demonstrable biochemical characteristic indicates a substantially increased requirement and utilization of this substance to potentiate these various host resistance factors. The results of a clinical trial are presented in which 100 terminal cancer patients were given supplemental ascorbate as part of their routine management. Their progress is compared to that of 1000 similar patients treated identically, but who received no supplemental ascorbate. The mean survival time is more than 4.2 times as great for the ascorbate subjects (more than 210 days) as for the controls (50 days). Analysis of the survival-time curves indicates that deaths occur for about 90% of the ascorbate-treated patients at one-third the rate for the controls and that the other 10% have a much greater survival time, averaging more than 20 times that for the controls. The results clearly indicate that this simple and safe form of medication is of definite value in the treatment of patients with acvanced cancer.
TL;DR: VIP, at present a gastrointestinal hormone candidate, appears to represent a new neuronal peptide occurring in both the central and peripheral nervous system.
Abstract: The localization of the vasoactive intestinal polypeptide (VIP) has been studied with immunohistochemistry and radioimmunoanalysis. VIP immunoreactivity is present in gastrointestinal nerves, which constitute a quantitatively important nerve population that may be intrinsic to the gut wall. VIP-immunoreactive neurons are also found within the ventromedial hypothalamus and give off processes that travel latteral to the third ventricle. Results of radioimmunoanalysis strongly indicate that the immunoreactive material represents true VIP. Thus VIP, at present a gastrointestinal hormone candidate, appears to represent a new neuronal peptide occurring in both the central and peripheral nervous system.
TL;DR: An X-linked mutant, dunce, has been isolated that fails to display this learning in spite of being able to sense the odorant and electric shock and showing essentially normal behavior in other respects.
Abstract: Normal Drosophilia learn to avoid an odorant associated with electric shock. An X-linked mutant, dunce, has been isolated that fails to display this learning in spite of being able to sense the odorant and electric shock and showing essentially normal behavior in other respects.
TL;DR: Silver grains indicative of the binding of labeled [3H]diprenorphine are discretely localized in numerous areas of the brain with very high densities in the locus coeruleus, the substantia gelatinosa of the spinal cord, and in clusters within the caudate-putamen, amygdala, and parts of the periventricular gray matter.
Abstract: Opiate receptor sites in rat brain can be labeled in vivo by [3H]diprenorphine, a potent opiate antagoinst. Using techniques to minimize diffusion in fresh, frozen, unfixed brain, we have localized [3H]diprenorphine by autoradiography to visualize the distribution of opiate receptors. Silver grains indicative of the binding of labeled [3H]diprenorphine are discretely localized in numerous areas of the brain with very high densities in the locus coeruleus, the substantia gelatinosa of the spinal cord, and in clusters within the caudate-putamen, amygdala, and parts of the periventricular gray matter.
TL;DR: The structure has been determined and shown to be identical to the sequence of carboxyl-terminal 31 amino acids of ovine beta-lipotropin, and the peptide possesses very low lipotropic activity but significant opiate activity.
Abstract: The isolation of an untriakontapeptide from camel pituitary extracts has been described. Its structure has been determined and shown to be identical to the sequence of carboxyl-terminal 31 amino acids of ovine beta-lipotropin. The peptide possesses very low lipotropic activity but significant opiate activity.
TL;DR: A new autosomal recessive mouse mutation,Purkinje cell degeneration (pcd), is described, which results from postnatal degeneration of virtually all cerebellar Purkinje cells and progressing rapidly over the next 2 weeks.
Abstract: A new autosomal recessive mouse mutation, Purkinje cell degeneration (pcd), is described Mutants exhibit a moderate ataxia beginning at 3 to 4 weeks of age The ataxia results from postnatal degeneration of virtually all cerebellar Purkinje cells beginning around 15 to 18 days of age and progressing rapidly over the next 2 weeks In addition to the cerebellar disease there is slow progressive degeneration in the retina (photoreceptor cells) and olfactory bulb Also, adult males have abnormal sperm
TL;DR: An inhibitory activity for an erythrocyte in termediate bearing the properdin (P)-stabilized amplification C3 convertase, PC3bBb, was recognized in whole normal human serum and separated from C3b inactivator by its distinct physicochemical and functional characteristics.
Abstract: An inhibitory activity for an erythrocyte in termediate bearing the properdin (P)-stabilized amplification C3 convertase, PC3bBb, was recognized in whole normal human serum and separated from C3b inactivator by its distinct physicochemical and functional characteristics. The inhibitory activity was found to reside in a protein that was purified to homogeneity and elicited a monospecific antibody in a rabbit. This protein was identified as beta1H and found to have a serum concentration of 516 +/- 89 mug/ml (mean +/- 1 SD). beta1H produced a dose related, first-order loss of convertase function and release of 125I-Bb from the P-stabilized intermediate, indicating a mechanism of action by decay-dissociation of Bb from the complex, PC3bBb. beta1H exhibited only a limited capacity to accelerate decay of C3bBb sites stabilized with C3 nephritic factor or to release 125I-Bb from such sites. Amplification of C3 cleavage by C3bBb may well determine whether initial complement activation by the classical or alternative activating sequence is beneficial or detrimental to the host. Regulation of this amplifying function is now recognized to occur at at least three steps: intrinsic decay which reflects the inherent lability of the C3bBb convertase; extrinsic decay-dissociation of Bb which is mediated by the effect of beta1H; and inactivation of exposed C3b by C3b inactivator. The stabilization of C3bBb by activated properdin minimizes intrinsic decay and protects C3b in the bimolecular complex from C3b inactivator. beta1H restores control of the system by decay-dissociation of the bimolecular complex, therby exposing C3b to C3b inactivator whose irreversible action prevents regeneration of the convertase at that site.
TL;DR: Serum stimulates the production of prostaglandins by transformed mouse fibroblasts and Hydrocortisone (cortisol) inhibits this stimulation, which may be the mechanism for the anti-inflammatory action of corticosteroids.
Abstract: Serum stimulates the production of prostaglandins by transformed mouse fibroblasts Hydrocortisone (cortisol) inhibits this stimulation The half-maximal inhibition occurs at 6x10-9 M Studies with cells labeled with [3H]arachidonic acid in their lipids show that the stimulation by serum results in the release of arachidonic acid from the cellular lipids, mostly phospholipids Hydrocrotisone inhibits this release but does not inhibit the production of prostaglandins from exogenously supplied arachidonic acid This inhibition of arachidonic acid release from phospholipids may be the mechanism for the anti-inflammatory action of corticosteroids
TL;DR: Although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging.
Abstract: Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging.
TL;DR: Together, both experiments indicate that the exogenous M-MuLV can be converted to an endogenous virus after infection of preimplantation embryos and that M- MuLV integrated into the germ line at one out of two possible integration sites.
Abstract: Mice were infected with the exogenous Moloney leukemia virus (M-MuLV) at two different stages of development. Either newborn mice (which can be considered as essentially fully differentiated animals) or preimplantation mouse embryos (at the 4-8 cell stage) were infected with M-MuLV. In both cases, animals that had developed an M-MuLV-induced leukemia were obtained. Two lines of evidence indicate that infection of preimplantation embryos, in contrast to infection of newborns, can lead to integration of the virus into the germ line. 1. Viremic males of the first backcross generation (N-1 generation) transmitted the virus to 50% of their offspring (N-2 generation) when mated with uninfected females. Likewise, a 50% transmission was observed from viremic N-2 and N-3 males to the next generations. 2. Molecular hybridization experiments revealed that viremic N-1 and N-2 animals carried one copy of M-MuLV per diploid mouse genome equivalent in all "non-target" organs tested. Together, both experiments indicate that the exogenous M-MuLV can be converted to an endogenous virus after infection of preimplantation embryos. The available evidence suggests that M-MuLV integrated into the germ line at one out of two possible integration sites. Thus, viremic backcross animals are heterozygous for a single Mendelian locus carrying the M-MuLV gene. During leukemogenesis an amplification of the M-MuLV from one copy to a maximum of four copies per diploid mouse genome equivalent takes place in the tumor tissues.
TL;DR: In this article, the authors studied cells dispersed with proteolytic enzymes from rheumatoid arthritic synovectomy specimens to determine the cell type(s) responsible for joint destruction, and found that 10-50% of these cells adhered to culture dishes within 24 hours and were of two main types: small, round cells and larger, stellate cells.
Abstract: We have studied cells dispersed with proteolytic enzymes from rheumatoid arthritic synovectomy specimens to determine the cell type(s) responsible for joint destruction. Initially 10-50% of these cells adhered to culture dishes within 24 hr and were of two main types: small, round cells and larger, stellate cells. During 1-4 days of culture, 5-25% had Fc receptors and 25-50% showed brisk phagocytosis. Daily producition, per 10(6) cells of collagenase (EC 3.4.24.3) (after trypsin pretreatment) was up to 70 mug of collagen fibrils lysed per min at 37 degrees (70 units), of prostaglandin (PGE2), up to about 1200 ng, and of lysozyme, up to about 100 mug. Under identical conditions of assay, fibroblasts grown from explants of synovium produced no detectable collagenase or lysozyme, and PGE2 was only 2-4 ng. With the dispersed cell preparations, macrophage markers (Fc receptors and lysozyme) were undetectable after 4 days and PGE2 decreased rapidly after about 7 more days. However, collagenase production continued for 3-25 weeks, and in some cultures, after cell passage. At these later stages, large, slow-growing stellate cells were predominant and could phagocytose carbon particles if incubated for greater than 6-8 hr. Indomethacin (14 muM) inhibited PGE2 but stimulated collagenase production whereas dexamethasone (10 nM) inhibited both. Production of PGE2 and collagenase in large amounts in vitro by these cells suggests that they may be involved in joint destruction in vivo. The precise origin of these synovial cells and the mechanisms responsible for the sustained production of collagenase at a high rate remain unidentified.
TL;DR: Cytological and genetic evidence shows that in this mutant the events associated with zygote formation are normal until the point of nuclear fusion, and the defect kn kaf1-1 appears to be nuclear limited.
Abstract: A mutant unable to fuse nuclei during mating has been isolated from standard wild-type Saccharomyces cerevisiae. Tetrad analysis of the mutation responsible for this defect (kar1-1) shows that it segregates as a single Mendelian factor. The defect kn kaf1-1 appears to be nuclear limited. Cytological and genetic evidence shows that in this mutant the events associated with zygote formation are normal until the point of nuclear fusion. The consequence of this defect is the formation of a multinucleate zygote which in subsequent divisions can segregate heterokaryons and haploid heterplasmons.
TL;DR: The results are consistent with a role for CSP in cell adhesion and morphology but not in growth control, and the morphological changes are antagonized by antibody to CSP.
Abstract: We have isolated the major cell surface glycoprotein of chick embryo fibroblasts, CSP, and added it to a variety of transformed cells in vitro. The transformed cells become more elongated, often more flattened, and show increased adhesion to the substratum. Several transformed cell lines also align in striking parallel arrays. This alignment is characterized by a decrease in the amount of nuclear overlapping, probably indicating restoration of contact inhibition of movement. The morphological changes are antagonized by antibody to CSP. These effects of CSP are not associated with an elevation of cellular 3':5'-cyclic AMP. Moreover, the morphological reversion is not accompanied by an alteration in growth properties. Our results are consistent with a role for CSP in cell adhesion and morphology but not in growth control.
TL;DR: In cell culture a kidney epithelial cell line MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions, which has transport and permeability qualities of in vivo transporting epithelia.
Abstract: In cell culture a kidney epithelial cell line MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia. The cell layer can be readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms-cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCl, of 1.7. Net water flux, apical to basolateral, was 7.3 mul cm-2 hr-1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potential use of a homogeneous cell population in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels.
TL;DR: It is revealed that hydrogen evolution is a general phenomenon associated with nitrogen fixation by many nodulated nitrogen-fixing symbionts and that the extent of hydrogen evolution during nitrogen reduction is a major factor affecting the efficiency of nitrogen fixation in many agronomically important legumes.
Abstract: Nitrogenase-dependent hydrogen evolution from detached legume nodules and from reaction mixtures containing cell-free nitrogenase has been well established, but the overall effect of hydrogen evolution on the efficiency of nitrogen fixation in vivo has not been critically assessed. This paper describes a survey which revealed that hydrogen evolution is a general phenomenon associated with nitrogen fixation by many nodulated nitrogen-fixing symbionts. An evaluation of the magnitude of energy loss in terms of the efficiency of electron transfer to nitrogen, via nitrogenase, in excised nodules suggested that hydrogen production may severely reduce nitrogen fixation in many legumes where photosynthate supply is a factor limiting fixation. With most symbionts, including soybeans, only 40-60% of the electron flow to nitrogenase was transferred to nitrogen. The remainder was lost through hydrogen evolution. In situ measurements of hydrogen evolution and acetylene reduction by nodulated soybeans confirmed the results obtained with excised nodules. In an atmosphere of air, a major portion of the total electron flux available for the reduction of atmospheric nitrogen by either excised nodules or intact nodulated plants was utilized in the production of hydrogen gas. Some non-leguminous symbionts, such as Alnus rubra, and a few legumes (i.e., Vigna sinensis) apparently have evolved mechanisms of minimizing net hydrogen production, thus increasing their efficiency of electron transfer to nitrogen. Our results indicate that the extent of hydrogen evolution during nitrogen reduction is a major factor affecting the efficiency of nitrogen fixation by many agronomically important legumes.
TL;DR: A definitive test for developmental totipotency of mouse malignant teratocarcinoma cells was conducted by cloning singly injected cells in genetically marked blastocysts, indicating that a near-normal chromosome complement is sufficient for total restoration of orderly gene expression in a normal embryonic environment.
Abstract: A definitive test for developmental totipotency of mouse malignant teratocarcinoma cells was conducted by cloning singly injected cells in genetically marked blastocysts. Totipotency was conclusively shown in an adult mosaic female whose tumor-strain cells had made substantial contributions to all of the wide range of its somatic tissues analyzed; the clonally propagated cell lineage had therefore differentiated in numerous normal directions. The test cells were from "cores" of embryoid bodies of a euploid, chromosomally male (X/Y), ascites tumor grown only in vivo by transplantation for 8 years. The capacity of cells from the same source to differentiate, in a phenotypic male, into reproductively functional sperms, has been shown in our previous experiments [(1975) Proc. Nat. Acad. Sci. USA 72, 3585-3589]. Cells from this transplant line therefore provide material suitable for projected somatic and germ-line genetic analyses of mammalian differentiation based on "cycling" of mutation-carrying tumor cells through developing embryos. In some animals obtained from single-cell injections tumor-derived cells were sporadically distributed in developmentally unrelated tissues. These cases can be accounted for by delayed and haphazard cellular integration, and by a marked degree of sustained cellular developmental flexibility in early mammalian development, irrespective of certain classical "germ-layer" designations. All mosaic mice obtained have thus far been free of teratomas. In one case, the injected stem cell contributed only to the pancreas and gave rise to a malignancy resembling pancreatic adenocarcinoma. The high modal frequency of euploidy in these individually tested cells thus tends to indicate that a near-normal chromosome complement is sufficient for total restoration of orderly gene expression in a normal embryonic environment; it may also be necessary for teratoma stem-cell proliferation to be terminated there.
TL;DR: The results demonstrate the existence of two different electron transfer systems from NADPH to deoxyribonucleotides and provide a function for glutathione in DNA synthesis in E. coli.
Abstract: E. coli B tsnC 7004, an E. coli B/1 mutant with normal phenotype unable to replicate phage T7 DNA [Chamberlin, M. (1974)J. Virol. 14,509-516], contained no detectable level of thioredoxin when assayed with ribonucleotide reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Gently lysed E. coli tsnC 7004 cell extracts reduced CDP when supplemented with NADPH as efficiently as the parent strain E. coli B/1 despite the lack of thioredoxin, indicating the presence of another hydrogen transport system. This could be divided into two parts by heat treatment at 85degrees; one heat-stable fraction, which was active in the presence of dithiothreitol or glutathione, and one heat-labile fraction. Addition of yeast glutathione reductase [NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2] to the heated extracts restored full activity. The results demonstrate a novel hydrogen transport system in E. coli consisting of NADPH, glutathione, glutathione reductase, and a heat-stable enzyme called "glutaredoxin". Reduced glutathione at physiological concentrations functions as hydrogen donor for ribonucleotide reduction only in the presence of glutaredoxin. Glutaredoxin was not reduced by E. coli thioredoxin reductase (NADPH:oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) and showed no crossreaction with antibodies against thioredoxin. These results demonstrate the existence of two different electron transfer systems from NADPH to deoxyribonucleotides and provide a function for glutathione in DNA synthesis.