bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose binding and transport proteins extracted from fast-growing chicken fibroblasts
Authors: Lee Sung G. , Lipmann Fritz
Published: May 2006

Preconfluent or confluent fibroblasts grown in 5% serum medium yielded, without cell lysis, all the glucose-binding protein and most of the transport-stimulating activity in the cell wash fluid obtained with a 10 mM sodium octanoate-containing solution. For assay, octanoate was removed, and after the binding protein was labeled with [
14
C]glucose, the factors were chromatographed on Sephadex G-200 and the transport-stimulating and factor-bound [
14
C]glucose activities were measured. Three peaks were separated, which more or less overlapped for both functions; upon chromatography on DEAE-cellulose, these peaks yielded overlapping or separate peaks for the two functions, presumably indicating their separability. Serum, when similarly chromatographed, showed only peaks for transport which, with the exception of one major peak with both functions, more or less overlapped with those from the wash fluid. Glucose transport rates, when compared in fibroblasts grown in glucose and in fructose and in Rous sarcoma virus-transformed cells grown in glucose, were in the proportion of 1:3.7:6.3. Addition of extracted transport protein stimulated the transport of both the glucose-grown and fructose-grown normal cells but showed no effect on the transport of transformed cells. Addition of transport protein induced the formation of [
14
C]deoxyglucose 6-phosphate in amounts proportional to the increased transport of [
14
C]deoxyglucose into fibroblasts. On sodium dodecyl sulfate electrophoresis, using the tightly bound [
14
C]glucose for assay, purified binding protein yielded large fractions of 36,000 and 18,000 and small ones of 55,000 and 73,000 daltons; the 18,000-dalton fraction is supposedly the monomeric form of the binding protein.
Introduction of antigenic phospholipids into the plasma membrane of mammalian cells: organization and antibody-induced lipid redistribution.
Authors: Schroit A J , Pagano R E
Published: May 2006

Phosphatidylethanolamine bearing the 2,4,6-trinitrophenyl hapten was introduced into the surface membrane of mammalian fibroblasts by incubating the cells with small unilamellar vesicles containing this hapten-conjugated lipid. Consistent with integration of the antigen into the plasma membrane lipid bilayer, the exogenously supplied lipid was observed by immunofluorescence to diffuse rapidly (D greater than or equal to 0.6 X 10(-8) cm2/sec) over the surface of polykaryons formed between vesicle- and non-vesicle-treated cells. Association of the exogenous lipids with cells via adsorption of vesicles to the plasma membrane was rigorously excluded by a combination of ultrastructural and immunofluorescence studies. The distribution of the integrated antigenic lipid in the plasma membranes of vesicle-treated cells was followed by immunofluorescence microscopy. The exogenously supplied hapten-conjugated phospholipid was observed to be uniformly distributed and remained so for up to 1 hr at 37 degrees C. However, upon the addition of bivalent, but not monovalent, antihapten antibodies, the phospholipid underwent a rapid temperature-dependent redistribution, forming small patches that eventually coalesced into one or more large aggregates. This unexpected finding is discussed in terms of the mode of insertion of the lipid into the cell surface and the possible mechanisms by which bivalent ligands might alter the mobility and distribution of cell surface phospholipids.
Infectious murine leukemia virus from DNA of virus-negative AKR mouse embryo cells.
Authors: Lowy D R
Published: May 2006

DNA from virus-negative AKR mouse embryo cells is infectious for NIH 3T3 cells if the transfected cells are treated with 5-iododeoxyuridine (IdUrd) either before or after addition of the DNA. The virus isolated after transfection of the AKR DNA is an ecotropic murine leukemia virus (MuLV) indistinguishable from endogenous AKR MuLV. The AKR DNA is not infectious in the absence of IdUrd treatment of the recipient cells. DNA from AKR cells treated with IdUrd before DNA isolation is not infectious unless the recipient cells have been treated with IdUrd. Transfected DNA from NIH mouse cells is not infectious even when the recipient cells have been treated with IdUrd. DNA from cells chronically infected with AKR MuLV or Rauscher MuLV is infectious with or without IdUrd treatment of the recipient cells, but the infectivity is not enhanced by IdUrd. The results indicate that the endogenous AKR MuLV DNA genome is potentially infectious. The data are consistent with an IdUrd-sensitive restriction in the recipient NIH 3T3 cells that prevents viral replication from endogenous AKR MuLV DNA but does not prevent viral replication from MuLV DNA of productively infected cells.
Indirect estimates of mutation rates in tribal Amerindians.
Authors: Neel J V , Rothman E D
Published: May 2006

Three different formulations have been used to estimate the average rate/locus/generation with which mutation results in electrophoretically detectable variants of 28 proteins in 12 tribal Amerindian populations. All methods are indirect--i.e., they assume a reasonable approximation to equilibrium between mutation and loss of mutants from the population--and are based on the further assumption that the biochemical traits under consideration are essentially neutral in their phenotypic effects. Despite the fact that the methods draw on somewhat different aspects of the available data, there is satisfactory agreement between them, the average of the three estimates being 1.6 X 10(-5)/locus/generation. This average does not encompass mutation that does not result in a change in electrophoretic mobility or that results in loss of enzyme activity. It is noteworthy that this estimate is in satisfactory agreement with a recent estimate by Neel and Thompson [Neel, J. V. & Thompson, E. A. (1978) Proc. Natl. Acad. Sci. USA 75, 1904--1908] of the mutation pressure necessary to maintain the number of "private" genetic polymorphisms being encountered in Amerindian tribes, if selection is not a factor.
Isolation of host-range variants of mouse mammary tumor viruses that efficiently infect cells
in vitro
Authors: Howard David K. , Schlom Jeffrey
Published: May 2006

Host-range variants of mouse mammary tumor virus (MMTV) have been isolated that have the ability to productively infect cells
in vitro
with high efficiency (at multiplicities of infection ≤1) and with extremely short latent periods to the production of
de novo
virus (as short as 4 days after infection). These variants of the highly oncogenic MMTV of RIII, C3H, and GR mice were obtained by serial virus passage in feline cells. The resultant variant stocks react in group-specific radioimmunoassays for the MMTV major external glycoprotein (gp52) and major internal protein (p28), possess a protein profile similar to that of wild-type MMTV, and contain a virion-associated DNA polymerase with a magnesium cation preference. Addition of dexamethasone and insulin to culture media enhances the titer of
de novo
MMTV to levels of approximately 10
10
particles per 75-cm
2
flask (containing 5 × 10
6
cells) per 24 hr. Variant stocks exhibit no evidence of contamination with either murine or feline type C retroviruses, as assayed by various techniques. The variants of MMTV derived from C3H and RIII mice exhibit differential host ranges that include the ability to productively infect feline, canine, bat, mink, murine, and human cells. Use of these MMTV host-range variants now facilitates the study of the complete replicative cycle of MMTV as well as an elucidation of the interaction of MMTV with various hormones, physical or chemical carcinogens, and tumor promoters in the initiation and promotion of mammary neoplasia.
Dose-dependent preferential binding of polycyclic aromatic hydrocarbons to reiterated DNA of murine skin cells in culture.
Authors: Shoyab M
Published: May 2006

The distribution of active metabolites of polycyclic aromatic hydrocarbons bound to reiterated or unique regions of murine DNA has been studied by a DNA-DNA renaturation technique. Murine skin cells were exposed to different doses of radioactive polycyclic aromatic hydrocarbons for 24 hr; then the hydrocarbon-labeled DNA was isolated, fragmented, and denatured. Renaturation kinetics and thermal stabilities of DNA-DNA duplexes were studied. At high carcinogen doses, polycyclic aromatic hydrocarbon adducts seem to be distributed equally among the DNA of all reiteration frequencies. At low carcinogen doses, however, a dose-dependent preferential binding to reiterated DNA sequences occurs. An inverse linear relationship appears to exist between the enrichment of hydrocarbon adducts in reiterated DNA sequences and the logarithm of the amount of total hydrocarbon bound to DNA.
Construction of a recombinant bacterial plasmid containing a chick pro-alpha2 collagen gene sequence.
Authors: Sobel M E , Yamamoto T , Adams S L , DiLauro R , Avvedimento V E , de Crombrugghe B , Pastan I
Published: May 2006

A recombinant plasmid containing chick pro-alpha2 collagen gene sequences has been constructed and cloned in Escherichia coli. Using partially purified collagen mRNA as template, we synthesized double-stranded DNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) from avian myeloblastosis virus and the Klenow A fragment of E. coli DNA polymerase I. From this complex mixture of double-stranded DNAs, a specific 200-base-pair restriction fragment was generated by cleavage with the restriction endonucleases BamHI and EcoRI. These enzymes also make unique cuts in the plasmid vector pBR322. The restriction fragment was inserted into pBR322 via these BamHI and EcoRI sites and cloned in E. coli chi1776. The cloned recombinant plasmid was shown to contain pro-alpha2 collagen DNA by its specific hybridization to chick pro-alpha2 collagen mRNA, as assayed in an in vitro translation system. Thus, a clone containing pro-alpha2 collagen DNA was constructed without first obtaining highly purified collagen mRNA.
Estrogen control of prolactin synthesis in vitro.
Authors: Lieberman M E , Maurer R A , Gorski J
Published: May 2006

Primary cultures of rat pituitary cells respond to estradiol-17beta by increased incorporation of radiolabeled precursors into prolactin but not into the bulk of other cellular proteins. The rate of increase in prolactin synthesis is dose dependent, reaching maximal levels in the physiological range of estradiol. At a concentration of 1 nM, estradiol, diethylstilbestrol, and estriol are stimulatory whereas androgens, progesterone, and corticosterone are without significant effect. Exposure of pituitary cells to 10 nM estradiol resulted in a 500% increase in prolactin synthesis after 7 days of culture. The results indicate that estradiol can stimulate prolactin synthesis through direct action on the pituitary.
Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor
Authors: Burstein David E. , Greene Lloyd A.
Published: May 2006

Studies on the mechanism of action of nerve growth factor (NGF) were carried out with PC12 rat pheochromocytoma cells. PC12 cells are uniquely useful for such studies because they respond to, but (unlike normal neurons) do not require, NGF and may undergo either generation or regeneration of neurites in response to NGF. Regeneration is defined here as NGF-dependent regrowth of neurites within 24 hr after subculture of NGF-treated PC12 cells. As in cultures of normal NGF-responsive neurons, neurite regeneration by PC12 cells occurs even in the presence of high concentrations of RNA synthesis inhibitors. Generation of neurites is defined as the
de novo
initiation of outgrowth when PC12 cells are exposed to NGF for the first time. In contrast to regeneration, neurite generation takes place with a lag of at least 24 hr and is blocked by low concentrations of RNA synthesis inhibitors. Such findings suggest that there are both RNA synthesis-dependent and -independent pathways in the mechanism whereby NGF stimulates neurite outgrowth. In addition, NGF-treated PC12 cells undergo a time-dependent loss of the capacity for neurite regeneration after pretreatment with RNA synthesis inhibitors or withdrawal of NGF. Such findings suggest that (
i
) initiation of neurite outgrowth requires NGF-stimulated, RNA synthesis-dependent accumulation of intracellular material(s), (
ii
) once such accumulation occurs, RNA synthesis-independent regeneration can occur (but only in the presence of NGF), and (
iii
) the turnover of such material(s) in the absence of their replacement leads to loss of the capacity for regeneration. A tentative sequence is presented for the events whereby NGF may stimulate neurite outgrowth.
G2 phase-specific proteins of HeLa cells.
Authors: Al-Bader A A , Orengo A , Rao P N
Published: May 2006

The objective of this study was to determine if HeLa cells irreversibly arrested in G2 phase of the cell cycle by a brief exposure to a nitrosourea compound were deficient in certain proteins when compared with G2-synchronized cells. Total cellular proteins of G2-synchronized, G2-arrested, and S phase-synchronized cells were compared by two-dimensional polyacrylamide gel electrophoresis. The S phase cells differed from the G2-synchronized and G2-arrested cells by the absence of about 35 and 25 protein spots, respectively, of a total of nearly 150. At least nine protein spots in the molecular weight range of 4--5 X 10(4) that were present in the G2-synchronized cells were absent in both the G2-arrested and the S phase cells. Thus, these studies suggest that the missing proteins are probably necessary for the transition of cells from G2 phase to mitosis. Supplying the missing proteins to the G2-arrested cells by fusion with G2-synchronized cells facilitated the entry of the former into mitosis.
DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes.
Authors: Rungger D , Türler H
Published: May 2006

Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and capsid proteins. Simian virus 40 large and small tumor antigens synthesized in the oocytes were indistinguishable, by gel electrophoresis and [35S]methionine-labeled tryptic peptide mapping, from the corresponding polypeptides synthesized in CV-1 African green monkey cells. The synthesis of large simian virus 40 tumor antigen implies the correct splicing of its mRNA, which is complementary to nonadjacent nucleotide sequences in the early region of the viral genome. Polyoma DNA directed synthesis of two polyoma tumor antigen polypeptides, 57,000 Mr and small tumor antigen, and of the main capsid protein.
Time-dependent resistance or susceptibility of tumor cells to cytotoxic antibody after exposure to a chemotherapeutic agent
Authors: Leibson P. J. , Schreiber H. , Loken M. R. , Panem S. , Rowley D. A.
Published: May 2006

We report that a chemotherapeutic agent (melphalan) can affect the sensitivity of tumor cells to cytotoxic antibody. Depending on the time interval between drug treatment and subsequent exposure to antibody and complement, the tumor cells can be either more resistant or more susceptible to antibody when compared to control cells. The number of tumor cells surviving the combined treatment was determined by a colony inhibition assay. The two antisera used in this study were directed against either virus-specific or myeloma protein-specific antigens on the surface of S107 murine myeloma cells; identical results were obtained with both sera. Twenty-four hours after exposure to the drug, the number of tumor cells surviving the antibody treatment increased. During this period of increased resistance, the tumor cells were temporarily arrested in the G
2
phase of the cell cycle. After this period of maximal resistance, the effect of cytotoxic antibody on the cells changed such that 4 days after melphalan treatment the cells were significantly more susceptible to the antibody than were the sham-treated control cells. The period of increased susceptibility correlated with an increased density of S107 myeloma protein and viral antigens on the surface of the tumor cells. Eight days after the drug treatment, the susceptibility of the tumor cells and the density of surface antigens both returned to normal levels. This study shows that the correct time interval between exposure to a drug and subsequent treatment with antibody is critical for maximal killing of the tumor cells. The basis for the differential sensitivity of the tumor cells to anti-body may be related to the drug-induced changes in the cell cycle and in antigen expression on the cell surface.
Normal human placentas contain RNA-directed DNA polymerase activity like that in viruses.
Authors: Nelson J , Leong J A , Levy J A
Published: May 2006

Extracts from over 100 normal human placentas have been examined for RNA-directed DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) activity. More than 80% of these placentas contained this enzyme activity, which banded at a density of 1.15-1.17 g/ml in sucrose. After heat treatment, this enzyme activity was shifted in density to 1.22-1.24 g/ml. The enzymatic activity was greater with (rC)n.(dG)12-18 than with (dC)n.(dG)12-18 and was not stimulated by (dG)12-18 alone. The product of the endogenous reaction, which was sensitive to RNase, had the characteristics of a small DNA associated with a large RNA by hydrogen bonding. Electron microscopic inspection of the material with a density of 1.15-1.17 g/ml revealed numerous retrovirus-like particles with central electron-dense cores and double-membraned envelopes. The enzyme may be associated with the retrovirus-lik particles noted in the trophoblast layer of some human placentas.
Competitive inhibition of benzodiazepine binding by fractions from porcine brain.
Authors: Colello G D , Hockenbery D M , Bosmann H B , Fuchs S , Folkers K
Published: May 2006

Fractions of porcine cerebral cortex extract separated by molecular weight on a Sephadex G-75 column were tested for their activities and potencies to inhibit [3H]benzodiazepine binding to rat brain homogenates. The fractions spanned molecular weights from 500 to 100,000. A potent inhibitor (benzodiazepine-competitive factor I, BCF-I) was discovered in the fraction containing substances with molecular weights from 40,000 to 70,000. Equilibrium binding studies indicated that BCF-I was a competitive inhibitor, making it a candidate as a benzodiazepine endogenous factor or profactor. BCF-I was heat stable, but trypsin digestion destroyed its activity. Another inhibitory fraction (BCF-II) was 1/5th as active as BCF-I and contained substances with molecular weights from 1000 to 2000.
Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.
Authors: Todaro G J , Sherr C J , Sen A , King N , Daniel M D , Fleckenstein B
Published: May 2006

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line.
Fluorine-19 nuclear magnetic resonance studies of Escherichia coli membranes.
Authors: Gent M P , Cottam P F , Ho C
Published: May 2006

Several fluorinated fatty acids of the general structure CH3(CH2)13--mCF2(CH2)m--2COOH are incorporated biosynthetically as unsaturated fatty acid analogues into the phospholipids of Escherichia coli. Under optimum conditions an unsaturated fatty acid autotroph, K1060B5, can be grown so that 50% of the total phospholipid fatty acids are 8,8-difluoromyristate. Conditions are found for which more than 20% of the fatty acids are fluorinated before a decrease in growth rate is observed. We have used 19F nuclear magnetic resonance to examine membranes isolated from E. coli grown under the latter conditions. A comparison is made with spectra of aqueous dispersions of extracted E. coli phospholipids and model multilayer phospholipid membranes. An explanation of the 19F resonance line shape in these membrane systems and the relationship to a molecular order parameter is given. It is apparent that 19F nuclear magnetic resonance is more sensitive to the degree of ordering or fluidity of phospholipids than spin labels or fluorescent probes. For instance, a dramatic effect of membrane protein on lipid fluidity can be seen. Finally, this method can be used to measure the proportion of frozen and fluid lipid in biological membranes at temperatures within the span of the gel-to-lipid phase transition.
Direct fluorescent assay of urokinase and plasminogen activators of normal and malignant cells: kinetics and inhibitor profiles.
Authors: Zimmerman M , Quigley J P , Ashe B , Dorn C , Goldfarb R , Troll W
Published: May 2006

A direct rate assay for plasminogen activator has been developed using a synthetic fluorogenic peptide substrate, 7-(N-Cbz-glycylglycylargininamido)-4-methylcoumarin trifluoroacetate. The assay correlates well with the standard 125I-labeled fibrin plate assay using highly purified urokinase, culture fluids from WI-38, Chinese hamster vary or HeLa cells, or Rous sarcoma virus-transformed chick fibroblasts as the source of plasminogen activator. The assay is sensitive, rapid, and linear throughout a wide range of enzyme concentrations. With this substrate it is possible to determine inhibitor profiles for the various plasminogen activators, independently of the interfering potential of plasmin. All of the enzymes tested are inhibited by leupeptin and antipain but not by the related aldehydes, elastatinal and chymostatin. The macromolecular inhibitors soybean trypsin inhibitor and trasylol have little or no effect on the plasminogen activators tested. This substrate should be useful for the study of the effect of various agents on functional changes in cells secreting this enzyme and also should allow kinetic measurements of potential inhibitors.
Isolation of the
in vivo
emitter in bacterial bioluminescence
Authors: Gast Robert , Lee John
Published: May 2006

A blue fluorescence protein has been isolated and purified from extracts of the luminous bacterium
Photobacterium phosphoreum
. It is a single polypeptide of molecular weight 22,000 with absorption maxima at 274 and 418 nm. It is efficiently fluorescent (ϕ

F

0.45), with a fully corrected spectral maximum (476 nm) and distribution identical to the
in vivo
bioluminescence from this same type of bacterium. At low concentration this fluorescence shifts towards the red and becomes identical to the
in vitro
bioluminescence emission. This spectral shift apparently results from a change in the protein pulled by dissociation of the chromophore (

K
d

[unk] 10
-7
M). If the blue fluorescence protein is included in the
in vitro
bioluminescence reaction with reduced FMN, oxygen, aldehyde, and luciferase (
P. phosphoreum
), the bioluminescence spectrum is shifted towards the blue from its maximum at 490 nm to one at 476 nm, where it is again identical in all respects to the
in vivo
bioluminescence spectrum. This is accompanied by an increase in the initial light intensity by an order of magnitude at saturating levels of blue fluorescence protein, and the specific light yield of the luciferase is increased 4-fold. It is suggested that the blue fluorescence protein acts as a sensitizer of the bacterial bioluminescence reaction.
Cotransfer of thymidine kinase and galactokinase genes by chromosome-mediated gene transfer.
Authors: McBride O W , Burch J W , Ruddle F H
Published: May 2006

The Chinese hamster genes for thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) and galactokinase (ATP:D-galactose 1-phosphotransferase, EC 2.7.1.6) have been cotransferred to mouse cells by chromosome-mediated gene transfer. Hamster metaphase chromosomes were incubated with mouse B82 cells and 22 independent colonies were isolated in a selective medium. All of the 12 colonies analyzed expressed the donor form of thymidine kinase; the hamster form of galactokinase was also expressed in 2 of these colonies, indicating cotransfer with a frequency of about 20%. There was coordinate loss of both transferred genes from each colony when selection was applied for the loss of thymidine kinase alone. Comparison of the regional localization of these two linked genes with the frequency of cotransfer suggests that the transgenome is probably not larger than about 0.25% of the donor genome.
Minichromosome from BK virus as a template for transcription in vitro.
Authors: Meneguzzi G , Pignatti P F , Barbanti-Brodano G , Milanesi G
Published: May 2006

BK virus DNA can be extracted from virions as a nucleoprotein complex containing about 20 nucleosomes. Transcription of this "minichromosome" with Escherichia coli RNA polymerase indicates that both initiation and elongation of RNA chains are reduced by the presence of nucleosomes. Hybridization analysis of RNA made on the complex shows preferential transcription of one region of BK virus genome. No increase in strand selection is observed with respect to transcription of purified superhelical BK virus DNA.
Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders
Authors: Mears J. Gregory , Ramirez Francesco , Leibowitz David , Nakamura Frank , Bloom Arthur , Konotey-Ahulu Felix , Bank Arthur
Published: May 2006

Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [
32
P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With
Eco
RI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained β-like gene sequences assayed with β globin cDNA probe. One β-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three β gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing β-like genes were absent from δβ thalassemic DNA and one new fragment containing β-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of β-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with δβ thalassemia.
Topographical analysis of regulatory and metal ion binding sites on glutamine synthetase from
Escherichia coli
:
13
C and
31
P nuclear magnetic resonance and fluorescence energy transfer study
Authors: Villafranca J. J. , Rhee S. G. , Chock P. B.
Published: May 2006

The paramagnetic effect of Mn(II) on
13
C and
31
P nuclear magnetic resonance signals from the [
2
-
13
C]ATP adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] from
Escherichia coli
was measured. This effect permitted the determination of distances from the 2-C position and the phosphorus of covalently bound AMP to the two Mn(II) binding sites, n
1
and n
2
. Binding of Mn(II) to the n
1
site converts an inactive apo-enzyme to its active form, while the metal ion bound at n
2
occupies the metal-nucleotide substrate site. The distances from Mn(II) at the n
1
and n
2
sites to phosphorus are ∼10 and ∼7 Å and to the 2-C position of the adenine ring are ∼12 and ∼11 Å, respectively. The fluorescence energy transfer method was used to determine distances between Co(II) at n
1
and n
2
and the adenylyl site. For this experiment the enzyme was adenylylated with ε-ATP. The distances between ε-adenine and Co(II) at n
1
and n
2
are ∼13 and ∼11 Å, respectively. Quantitation of the paramagnetic effect due to Co(II) on the
31
P nuclear magnetic resonance signal yielded values of 8 and 6 Å for the distances between the phosphorus of the covalently bound AMP and the n
1
and n
2
sites, respectively. The results reveal that the covalent modification site is very close to the catalytic center of the enzyme. In this study both nuclear magnetic resonance and fluorescence energy transfer techniques have been used to determine distances between the same set of sites on an enzyme surface.
Addition of mononucleotides to oligoribonucleotide acceptors with T4 RNA ligase.
Authors: Kikuchi Y , Hishinuma F , Sakaguchi K
Published: May 2006

RNA ligase induced by bacteriophage T4 catalyzed the addition of nucleoside 5',3'-diphosphates to oligoribonucleotide acceptors in the presence of ATP. The reactions proceeded in equimolar concentrations of donors and acceptors. Several oligonucleotides of defined sequence were synthesized by this method in yields of 28-68%. The enzyme required the phosphate ester at the 3' position of the donor molecule, nucleoside 5',2'-diphosphates being unable to serve as donors. Thymidine 5',3'-diphosphate was active as a donor for the enzyme. The ligation product, (Ap)2ApCp, was also obtained from the reaction of (Ap)2A and 5'-adenylylated cytidine 5',3'-diphosphate (A5'pp5'Cp) with RNA ligase in the absence of ATP. These results show that the minimal substrate for RNA ligase is a nucleoside 5',3'-diphosphate.
Inhibition by colchicine of changes in amino acid transport and initiation of DNA synthesis in regenerating rat liver.
Authors: Walker P R , Whitfield J F
Published: May 2006

The uptake of amino acids (measured with alpha-aminoisobutyric acid) by hepatocytes stimulated to proliferate by partial hepatectomy followed a biphasic pattern, with an initial peak 8-10 hr after surgery followed by a release of amino acids between 10 and 16 hr and a further increase between 16 and 20 hr after the operation. The second period of increased uptake coincided with the increase in DNA synthesis by the hepatocytes. Colchicine, and other microtubule disrupters administered at the time of partial hepatectomy, abolished the first period of increased amino acid uptake and prevented the cells from initiating DNA synthesis. Pulse-labeling experiments with alpha-aminoisobutyric acid revealed that colchicine did not inhibit amino acid transport per se, but prevented the increased capacity for amino acid transport induced by partial hepatectomy. An injection of colchicine 14 hr after hepatectomy prevented the release of amino acids by the liver and also substantially reduced the initiation of DNA synthesis. The data suggest that the microtubules may play a role in proliferative activation and also in the initiation of DNA synthesis of hepatocytes in vivo and that changes in amino acid transport are linked to these microtubule-requiring processes.
Lambda2 light chains in normal mouse immunoglobulins.
Authors: Blaser K , Eisen H N
Published: May 2006

Light (L) chains of the lambda type are rare in mouse immunoglobulins. One lambda chain, the L chain of myeloma protein 315 (L315), differs in amino acid sequence at many positions in the COOH-terminal domain from all other lambda chains whose sequences have been determined (called lambda1 chains). To determine whether chains of the L315 type (called lambda2) occur in normal mouse immunoglobulins, we synthesized the COOH-terminal peptides expected in tryptic digests of K, lambda1, and L315 and developed procedures to separate the S-carboxymethyl derivatives of these peptides. Peptide maps of tryptic digests of [14C]carboxymethyl-labeled L chains from normal serum immunoglobulins showed that about 1% of mouse 7S immunoglobulins have L chains of the L315 or lambda2 type.
Pseudotypes of feline sarcoma virus contain an 85,000-dalton protein with feline oncornavirus-associated cell membrane antigen (FOCMA) activity.
Authors: Sherr C J , Sen A , Todaro G J , Sliski A , Essex M
Published: May 2006

Feline sarcoma virus (FeSV) rescued from transformed nonproducer mink or rat cells contains two FeSV-specific antigens (p15 and p12), and the feline oncornavirus-associated cell membrane antigen (FOCMA). All three antigens are helper virus-independent and are encoded by the FeSV genome, FOCMA, p15, and p12 antigens cochromatograph as phosphorylated molecules of 85,000 molecular weight (pp85), adsorb to immunoadsorbant columns prepared with antibodies to feline leukemia virus (FeLV), and are precipitated with antisera to FeLV or FOCMA. Antibodies to FOCMA can be adsorbed with fractions containing pp85 but not with FeLV proteins, including p15 and p12. Thus, a virus-coded tumor antigen which immunizes cats against tumors induced by feline type C viruses is packaged in FeSV particles and is linked to viral structural protein.
Plasma radioiron kinetics in man: explanation for the effect of plasma iron concentration.
Authors: Skarberg K , Eng M , Huebers H , Marsaglia G , Finch C
Published: May 2006

The plasma iron turnover was measured in 19 normal subjects. A correlation was found between plasma iron concentration and plasma iron turnover. In addition to the turnover of 55Fe at normal plasma iron concentration (predominantly monoferric transferrin), a second turnover in which the labeled plasma was saturated with iron (to produce predominantly diferric transferrin) was studied with 50Fe. It was demonstrated that diferric transferrin had a greater rate of iron turnover but that the distribution between erythroid and non-erythroid tissues was unchanged. It was concluded that plasma iron turnover is dependent on the monoferric/diferric transferrin ratio in the plasma but that the internal distribution of iron is unaffected.
Serotonin and substance P coexist in neurons of the rat's central nervous system
Authors: Chan-Palay Victoria , Jonsson Gösta , Palay Sanford L.
Published: May 2006

5-Hydroxytryptamine (serotonin)-containing neurons in the rat's medullary raphe and interfascicularis hypoglossi cell groups were identified by means of autoradiography following prolonged intraventricular administration of 5-hydroxy[
3
H]tryptamine, fluorescence histochemistry for the demonstration of endogenous 5-hydroxytryptamine, and microspectrofluorimetric analysis of excitation and emission spectra. Immunocytochemical methods (the unlabeled primary antibody—peroxidase antiperoxidase and indirect immunofluorescence methods) were applied with antisera to substance P in order to localize immunoreactivity in these medullary neurons. It was demonstrated that the raphe nuclei and the interfascicularis hypoglossi nucleus are heterogeneous cell groups that contain: (
i
) Neurons that display both an uptake—storage capacity for 5-hydroxy[
3
H]tryptamine and a formaldehyde-induced fluorescence with spectral characteristics identical to those of the 5-hydroxytryptamine fluorophor. These cells exhibit high to low fluorescence intensities without detectable substance P-like immunoreactivity. (
ii
) Neurons with various 5-hydroxytryptamine fluorescence intensities and intense to low degrees of substance P-like immunoreactivity. (
iii
) Neurons with various degrees of substance P-like immunoreactivity without detectable 5-hydroxytryptamine fluorescence or 5-hydroxy[
3
H]tryptamine uptake and storage capacity. These results indicate that some neurons contain high or low levels of only 5-hydroxytryptamine or substance P, whereas other neurons contain both 5-hydroxytryptamine and substance P in various proportions. The present findings demonstrate the presence of two putative transmitters, a biogenic amine and a polypeptide, within the same neuron in the mammalian central nervous system.
Species-specific cellular DNA-binding proteins expressed in mouse cells transformed by chemical carcinogens.
Authors: Sen A , Todaro G J
Published: May 2006

Mouse cells transformed by DNA and RNA tumor viruses and by chemical carcinogens have been examined for the presence of specific DNA-binding proteins by DNA-cellulose chromatography. Using mouse DNA-cellulose we have obtained single-stranded DNA-binding proteins from two clones transformed by chemical carcinogens. Simian virus 40 transformants also have a DNA-binding protein [the tumor (T) antigen] that binds to mouse and human DNA with comparable affinity. Mouse sarcoma virus-transformed cells and two other chemically transformed clones showed no difference in DNA-binding protein pattern compared to the untransformed parental cell. The DNA-binding proteins isolated from the chemically transformed cell clones are between 25,000 and 30,000 daltons by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. These cellular "T proteins" bind to the homologous mouse cellular DNA with a higher affinity than to heterologous human cellular DNA.
Insulin and epidermal growth factor-urogastrone: Affinity crosslinking to specific binding sites in rat liver membranes
Authors: Sahyoun N. , Hock R. A. , Hollenberg M. D.
Published: May 2006

Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with
125
I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with
125
I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization.
Brillouin light scattering measurement of the elastic properties of aligned multilamella lipid samples
Authors: LePesant Jean-Pierre , Powers L. , Pershan P. S.
Published: May 2006

Brillouin measurements of the elastic properties are presented for aligned multilamella samples of both pure dipalmitoyl phosphatidylcholine at various temperatures and water concentrations and for the same compound containing 33 mol% cholesterol at various temperatures and two water concentrations. For pure dipalmitoyl phosphatidylcholine the elastic moduli change significantly at the gel transition and the modulus for area compression of individual bilayers is inferred to be an order of magnitude larger below the gel transformation than above. The presence of cholesterol is shown to influence the elastic behavior of dipalmitoyl phosphatidylcholine.
Production of autoinhibitory factors by mouse lymphoma cells in vitro and its relationship to thiol dependence.
Authors: Tanapat P , Gaetjens E , Broome J D
Published: May 2006

Five different kinds of mouse lymphoma cells conditioned culture medium so that it became inhibitory to thiol-dependent cells but not to thiol-independent cells. Conditioning was rapid and appeared to be complete in 1-3 hr. The inhibitory effects could be reversed by concentrations of 2-mercaptoethanol higher than those usually required for growth promotion. Inhibitory factors were concentrated by ultrafiltration and chromatography on Sephadex G-10 which showed their apparent molecular weights to be 300-600. The factors were stable at 100 degrees and to proteases but were inactivated (after reduction) by iodoacetamide, indicating that they may be thiols or disulfides.
Physical, morphological, and biochemical alterations in the membrane of AKR mouse cells after interferon treatment.
Authors: Chang E H , Jay F T , Friedman R M
Published: May 2006

Interferon treatment of AKR,C- cells was followed by the establishment of an antiviral state and apparently concomitant morphological, physical, and biochemical alterations of the cell plasma membrane. The density of the plasma membrane was significant altered, and the concentration of some plasma membrane glycoproteins and the number of intramembranous particles observed in freeze-fracture electron micrographs were increased. A parallel increase in the concentration of intramembranous particles and the resistance to viral infection during interferon treatment as well as their parallel decrease upon removal of interferon suggests a relationship between the particle density and the establishment of the antiviral state.
Sequence variation among heavy chains from inulin-binding myeloma proteins
Authors: Vrana Mark , Rudikoff Stuart , Potter Michael
Published: May 2006

The entire sequences of the variable region of four heavy chains from BALB/c inulin-binding myeloma proteins have been determined. Among the four proteins there are six amino acid differences, all of which occur in the framework portion of the variable region. All of the six amino acid substitutions can be explained by single base mutations at the DNA level. The pattern of diversity in these proteins is compared to a previously reported group of heavy chains from phosphorylcholine-binding myeloma proteins. Unlike the phosphorylcholine-binding proteins, which (with the exception of two that are identical) have size and sequence differences in their complementarity regions, the inulin-binding heavy chains all have identical complementarity regions with H3 being extremely short. The pattern of variation observed in the anti-inulin heavy chains appears to be most easily explained by a somatic mutation mechanism. However, because none of the substitutions occur in complementarity-determining regions, they presumably would have no selective advantage and would not alter binding specificity. These proteins have further been shown to have crossreacting antigenic determinants (idiotypes). Five of the six sequence differences observed occur at positions that are internal in the molecule and thus presumably would not account for the idiotypic differences. These results suggest that most of the observed idiotypic crossreactivities will be due to differences in the light chains of the anti-inulin proteins.
Differences in behavior and prognosis between leukemic and lymphomatous forms of a transplantable hamster lymphocytic neoplasm induced by simian virus 40.
Authors: Diamandopoulos G T
Published: May 2006

A lymphocytic leukemia of probable monoclonal derivation, induced in a Syrian golden hamster by the oncogenic DNA simian virus 40, was adapted to grow in the allogeneic host either as leukemia or as lymphoma. The leukemia, which was produced by transplanting subcutaneously neoplastic lymphocytes that had circulated through and/or proliferated in lymph nodes and spleen, was characterized by dissemination with systemic manifestations and poor prognosis. The lymphoma, which was produced by transplanting subcutaneously neoplastic lymphocytes that had proliferated at subcutaneous sites of cell implantation, was characterized by localization and favorable prognosis. Evidence indicates that the tissue environment the neoplastic lymphocytes encounter during circulation and/or proliferation regulates their subsequent behavior in the intact host. Since the leukemic and lymphomatous forms of this animal model resemble very closely the analogous human lymphocytic neoplasms, it can serve as a means to elucidate the factors responsible for the differences in their behavior and to determine how these differences may influence prognosis and response to therapy.
Coherent stimulated light emission (lasing) in covalently linked chlorophyll dimers
Authors: Hindman James C. , Kugel Roger , Wasielewski Michael R. , Katz Joseph J.
Published: May 2006

The covalently linked chlorophyll
a
dimer exhibits remarkably different properties in the folded and open configurations. In the folded configuration the absorption maximum is at 695 nm and the fluorescence maximum is at 730 nm. Laser output at 733 and 735 nm is obtained for solutions in wet benzene and 0.1 M ethanol/toluene, respectively. Measurements of fluorescence lineshapes, made with a transverse excited atmospheric (TEA) nitrogen laser for excitation, show the lifetime shortening associated with stimulated emission resulting from appreciable concentrations of molecules in S
1
excited states. In contrast, the open dimer has absorption and fluorescence spectra essentially the same as those of chlorophyll
a
monomer. Unlike either the folded dimer or chlorophyll
a
monomer, the open dimer shows no laser emission or fluorescene lifetime shortening. It does not appear that the behavior of the open dimer can be explained in terms of excimer or triplet formation or by nonradiative decay processes. It is suggested that absorption of the exciting radiation by S
1
, leading to the formation of an exciplex or charge transfer state, may be involved. Significantly, no large changes in fluorescence quantum yield or fluorescence lifetime are observed for these dimers as compared to monomer chlorophyll. This suggests that concentration quenching and lifetime shortening in condensed chlorophyll systems involve more than the simple proximity of two chlorophyll molecules.
RNA-dependent RNA polymerase of tobacco plants
Authors: Ikegami M. , Fraenkel-Conrat H.
Published: May 2006

Several properties of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) active fractions obtained from tobacco plants, be they uninfected, infected with tobacco mosaic virus, or infected with tobacco necrosis virus, were compared. By the seven criteria tested, the RNA-dependent RNA polymerase from these three sources behaved the same, although its activity is greatly but variably stimulated by the two virus infections. It thus appears probable that these two viruses do not code for this enzyme, but rely for their replication on their ability to stimulate production of a host enzyme. The conclusion that cells contain RNA replicating capability represents a modification of the central dogma of molecular biology.