bluuebunny/crossref_metadata_2025 huggingface dataset:
Accumulation of DNA strand breaks and methotrexate cytotoxicity.
Authors: Li J C , Kaminskas E
Published: May 2006

There was a progressive formation of strand breaks in mature DNA of Ehrlich ascites tumor cells that were treated with methotrexate. Cells were labeled with [14C]thymidine before incubation with methotrexate, and DNA strand breaks were measured by alkaline and by neutral filter elution methods. DNA single-strand breaks accumulated in a linear fashion as a function of time during the first 10 hr of incubation with 2 microM methotrexate. Thereafter, the accumulation of DNA strand breaks deviated from linearity because of progressive cell death. The extent of DNA strand breakage in cells that had been incubated with methotrexate for 24 hr was as high as in cells that had been irradiated with 300 rads. DNA strand breaks persisted in cells that were incubated, after exposure to methotrexate, in medium containing thymidine, hypoxanthine, and nonessential amino acids, indicating that these strand breaks were poorly repaired. Cell death commenced after 10 hr of incubation with methotrexate and continued during the following 3-4 days. These findings suggest that cell death was due to a lethal accumulation of DNA strand breaks. The formation of DNA strand breaks is probably due to inefficient DNA repair, resulting from the inhibition of syntheses of thymidylate and of purine nucleotides. The accumulation of DNA strand breaks was minimal in growth-arrested cells, which are resistant to methotrexate toxicity.
An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections.
Authors: Keller G A , Tokuyasu K T , Dutton A H , Singer S J
Published: May 2006

Ultrathin frozen sections are ideal substrates with which to carry out immunolabeling experiments in electron microscopy. However, the ultrastructural delineation in positively stained frozen sections has not been as detailed as in conventionally osmium-stained and plastic-embedded sections. We now describe a simple technique in which immunolabeled ultrathin frozen sections are subsequently treated with osmium tetroxide, dehydrated, and then embedded in plastic by impregnation with a monomer to the thickness of the section, followed by polymerization of the monomer. By this technique ultrastructural definition as good as that of conventional plastic sections is achieved, while the high density and specificity of immunolabeling characteristic of ultrathin frozen sections are retained.
Variants of the cell recognition site of fibronectin that retain attachment-promoting activity.
Authors: Pierschbacher M D , Ruoslahti E
Published: May 2006

A tetrapeptide sequence, Arg-Gly-Asp-Ser, is the minimal structure recognized by cells in the large, adhesive glycoprotein fibronectin. We now have defined the structural requirements for this cell recognition site by testing several synthetic variants of the active tetrapeptide sequence. The conservative substitutions of lysine for arginine, alanine for glycine, or glutamic acid for aspartic acid each resulted in abrogation of the cell attachment-promoting activity characteristic of the natural sequence. However, in the position of the serine residue, some alterations were compatible with activity. Assay of peptides containing the structure Arg-Gly-Asp-X (where X = another amino acid residue) showed that an Arg-Gly-Asp-Val sequence predicted to be present in some, but not all, fibronectin molecules as a result of alternative RNA splicings could potentially create a second cell attachment site in those fibronectin polypeptide chains carrying that sequence. Other proteins with potentially active Arg-Gly-Asp-X sequences include several proteins that are known to interact with the cell surface. Among these are various types of collagens, thrombin, and discoidin, a slime-mold protein that may be involved in cell aggregation. The result presented here show that the arginine, glycine, and aspartic acid residues are absolutely required for the cell recognition, and that the surrounding amino acids may play a role in the expression of cell attachment activity in fibronectin and other proteins having this sequence. We suggest, based on these data, that this recognition mechanism may be common to a number of biological systems.
Production and characterization of monoclonal antibodies with specificity for the S100 beta polypeptide of brain S100 fractions.
Authors: Van Eldik L J , Ehrenfried B , Jensen R A
Published: May 2006

S100 refers to a heterogeneous fraction of low-molecular-weight acidic calcium-binding proteins. We report here production and characterization of two mouse hybridomas that secrete monoclonal antibodies that appear to be specific for the S100 beta polypeptide of brain S100 preparations. By ELISA, RIA, and immunoblotting analysis, the monoclonal antibodies react specifically with S100 beta and show little or no reactivity with any S100 alpha-like polypeptides. In addition, there is no reactivity with the structurally homologous proteins calmodulin and troponin C. The utility of these monoclonal antibodies for immunocytochemical studies of clinical pathology specimens has been demonstrated by examination of S100 beta localization in human autopsy brain and anaplastic astrocytoma sections. S100 beta is localized primarily in glial cell cytoplasm and processes, with no specific staining observed in glial cell nuclei, erythrocytes, or neuronal cells. These monoclonal antibodies may have important applications in pathological examination of surgical specimens as a specific marker for tumors containing S100 beta, will allow a more precise interpretation of the distribution and localization of S100 beta in both normal and neoplastic tissues, and may provide insight into the physiological functions of the S100 proteins.
Human embryonic zeta-globin chains in adult patients with alpha-thalassemias.
Authors: Chung S W , Wong S C , Clarke B J , Patterson M , Walker W H , Chui D H
Published: May 2006

Human embryonic zeta-globin chains are alpha-globin-like chains that are normally present during the first three months of gestation. In this investigation, zeta-globin chains measured by a specific and sensitive radioimmunoassay and by an electrophoretic technique were found to be present in all 7 patients studied with hereditary Hb H disease, and in 8 out of 24 patients with alpha-thalassemia trait. zeta-Globin chains were not detected in 20 other patients with beta-thalassemia trait. These results suggest that the deletion of two alpha-globin genes on the same chromosome is accompanied by the continued expression of embryonic zeta-globin genes in adult individuals.
Purification and characterization of acidic fibroblast growth factor from bovine brain.
Authors: Thomas K A , Rios-Candelore M , Fitzpatrick S
Published: May 2006

Acidic brain fibroblast growth factor has been purified a minimum of 35,000-fold to apparent homogeneity by a combination of differential salt precipitation, ion exchange chromatography, gel filtration, isoelectric focusing, and hydrophobic chromatography on a C4 reversed-phase HPLC column. Two microheterogeneous forms of the molecule are obtained with apparent molecular masses of 16,600 and 16,800 daltons. The mitogen is highly active with half-maximal stimulation of BALB/c 3T3 fibroblasts at about 40 pg/ml in an assay using incorporation of [methyl-3H]thymidine into DNA.
Rapid release of galactose-terminated ligands after endocytosis by hepatic parenchymal cells: evidence for a role of carbohydrate structure in the release of internalized ligand from receptor.
Authors: Townsend R R , Wall D A , Hubbard A L , Lee Y C
Published: May 2006

We have compared the behavior of 125I-labeled asialoorosomucoid (ASOR) and 125I-labeled asialotriantennary glycopeptide (TRI) from alpha-1-protease inhibitor in the perfused rat liver. These two ligands are recognized and internalized by the Gal/GalNAc receptor system of hepatocytes. We found that both ligands could be efficiently degraded when continuously perfused through a liver at 37 degrees C. However, as much as 51% of the internalized 125I-labeled TRI was rapidly (t1/2 = 3 min) released intact into the perfusion medium under conditions that permitted ligand-receptor association. In contrast, ASOR was totally degraded under these conditions. When dissociation was promoted (i.e., in the presence of GalNAc or EGTA), as much as 57% of the internalized ASOR was rapidly released (also intact). In each case, the shorter the initial internalization phase, the greater the proportion of ligand that was subsequently released. Neither the degree of receptor occupancy nor the temperature (16-37 degrees C) during the initial internalization phase influenced the rate or extent of the release process. Electron microscopic localization of internal ASOR-horseradish peroxidase conjugate demonstrated that peripheral endosomes were the major compartment labeled during exocytosis. We conclude that substantial fractions of both ligands rapidly return to the cell surface after internalization, ASOR bound to receptor and most of TRI free from receptor. The remaining ligand molecules enter the lysosomal pathway.
Injection of mice with antibody to interferon renders peritoneal macrophages permissive for vesicular stomatitis virus and encephalomyocarditis virus.
Authors: Belardelli F , Vignaux F , Proietti E , Gresser I
Published: May 2006

Vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) multiply in only a small percentage of peritoneal macrophages freshly explanted from 4- to 6-week-old male or female DBA/2, BALB/c, C3H, C57BL/6, or Swiss mice. However, when these mice were injected intraperitoneally with potent sheep (or goat) anti-mouse interferon alpha/beta globulin 4 days prior to harvesting peritoneal macrophages, the viruses multiplied to high titers and most of the cells were infected, as determined by total virus yield (VSV and EMCV), percentage of VSV antigen-positive cells (immunofluorescence), and determination of VSV infectious centers. This effect was not observed when mice were inoculated with other sheep hyperimmune or normal serum globulins. Anti-interferon globulin appeared to act in vivo because incubation of this globulin with peritoneal macrophages during the period of cell attachment or during the 18 hr after virus absorption did not render these cells permissive for VSV. Injection of mice with anti-interferon globulin did not affect the binding and uptake of labeled VSV by peritoneal macrophages. Although the underlying mechanism of this phenomenon is unknown, the results suggest that there may be low levels of endogenous interferon that contribute to host defense by maintaining some cells in an antiviral state.
Molecular cloning and characterization of DNA sequences encoding rat and human atrial natriuretic factors.
Authors: Zivin R A , Condra J H , Dixon R A , Seidah N G , Chrétien M , Nemer M , Chamberland M , Drouin J
Published: May 2006

A cDNA copy of the message encoding rat atrial natriuretic factor (ANF) has been cloned in Escherichia coli, and its nucleotide sequence was determined. ANF appears to be synthesized as a larger precursor, atrial pronatriodilatin. The cDNA has an open reading frame potentially encoding a protein of 152 amino acids, of which the first 24 amino acids strongly resemble a signal sequence. This is followed by a sequence with 80% homology to a second vasoactive protein, porcine cardiodilatin. The ANF peptide is contained in the COOH-terminal portion of the protein. The DNA sequence corresponding to human ANF is also presented and displays a high degree of homology to its rat counterpart. These data provide further evidence for the expression in cardiac atria of a multifactor system that may contribute to the regulation of blood pressure and extracellular fluid volume.
Human apolipoprotein C-II: complete nucleic acid sequence of preapolipoprotein C-II.
Authors: Fojo S S , Law S W , Brewer H B
Published: May 2006

Apolipoprotein (apo) C-II is a cofactor for lipoprotein lipase, the enzyme that catalyzes the hydrolysis of triglycerides on plasma triglyceride-rich lipoproteins. The complete coding sequence of apoC-II mRNA has been determined from an apoC-II clone isolated from a human liver cDNA library. A 17-base-long synthetic oligonucleotide based on amino acid residues 5-10 of apoC-II was utilized as a hybridization probe to select recombinant plasmids containing the apoC-II sequence. Two thousand four hundred clones were screened and one apoC-II cDNA clone containing 500 bases was identified. DNA sequence analysis of this clone revealed a 101 amino acid C-II apolipoprotein containing a 22 amino acid signal peptide attached to the amino terminus of the 79 amino acid residue plasma apoC-II. The amino acid sequence of apoC-II determined by nucleic acid analysis is in agreement with the recently determined sequence of plasma apoC-II isolated from normal subjects. The determination of the complete cDNA sequence of apoC-II and the availability of a cDNA probe of apoC-II will facilitate our analysis of the biosynthesis and processing as well as the genomic organization of apoC-II in normal subjects and patients with dyslipoproteinemias characterized by hypertriglyceridemia.
Plasmid P1 replication: negative control by repeated DNA sequences.
Authors: Chattoraj D , Cordes K , Abeles A
Published: May 2006

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pieces of the incA fragment that each contain only three repeats destabilize P1 plasmids efficiently. This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro.
A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration.
Authors: Donehower L A , Varmus H E
Published: May 2006

We have used site-directed mutagenesis of cloned Moloney murine leukemia virus (MuLV) DNA to define a function encoded in the 3' region of the viral pol gene and required for efficient integration of viral DNA. One mutant, MuLV-SF1, contained a single base substitution (C to T at base 4950) that resulted in an arginine to cysteine change in a region highly conserved among retroviruses. Mutant DNA, introduced into rat cells by cotransfection with a herpes simplex virus thymidine kinase gene (HSV tk), directed production of virus particles with reverse transcriptase activity. Infection of cells with these particles led to synthesis of full-length linear and circular forms of unintegrated viral DNA; however, integrated viral DNA was decreased at least by a factor of 10 when examined by DNA hybridization, and the mutant particles were less efficient then wild-type virus at establishing an infection by a factor of at least 300. Pseudotypes formed with the proteins of MuLV-SF1 and the genome of a replication defective marker MuLV, carrying the HSV tk gene, were less effective by at least a factor of 100 in producing tk+ colonies than pseudotypes formed with proteins encoded by wild-type virus. When the MuLV-SF1 pseudotypes did produce tk+ cells, most of the proviruses were integrated aberrantly. We conclude that the MuLV-SF1 pol gene is defective for a function that is required for normal integrative recombination and dissociable from DNA synthesis.
Biochemical evidence that vesicles are the source of the acetylcholine released from stimulated PC12 cells.
Authors: Melega W P , Howard B D
Published: May 2006

Treatment of PC12 cells with AH5183 at concentrations of 40 nM to 40 microM inhibited the loading of newly synthesized acetylcholine into storage vesicles, but it had little effect on choline uptake, acetylcholine synthesis, or the vesicular content of previously loaded acetylcholine. AH5183 at 4 microM inhibited the Ba2+-evoked release of newly synthesized acetylcholine but not of older stores of acetylcholine. These data indicate that the vesicles are the source of the acetylcholine released from stimulated cells. AH5183 had little effect on the vesicular loading of dopamine and on the evoked release of dopamine, but at concentrations of 4-40 microM it did inhibit dopamine uptake by the cells.
Isolation of a large cholecystokinin precursor from canine brain.
Authors: Eysselein V E , Reeve J R , Shively J E , Miller C , Walsh J H
Published: May 2006

Cholecystokinin (CCK)-like immunoreactivity (CCK-LI) in a pool of 12 dog brains was extracted sequentially into boiling water and cold 2% trifluoroacetic acid. Gel filtration on Sephadex G-50 revealed three main molecular forms detected by a carboxyl-terminal antibody; one was eluted in the position of CCK-58 (58 amino acid residues long); a second, in the position of CCK-8; and a third, near the radioactive iodide marker. When the CCK-LI was purified by affinity chromatography using carboxyl-terminal CCK antibody followed by three steps of reversed-phase high-pressure liquid chromatography, three components were isolated and characterized by sequence microanalysis. The smallest component was the pentapeptide common to gastrin and CCK. The second peak was eluted in the same region as synthetic CCK octapeptide, and sequence analysis showed that the chemical structure of this biologically active region of canine CCK is identical to that found in sheep and pig brains. The 22-residue amino-terminal sequence of brain CCK-58 was: Ala-Val-Gln-Lys-Val-Asp-Gly-Glu-Pro-Arg-Ala-His-Leu-Gly -Ala-Leu-leu-Ala-Arg-Tyr-Ile-Gln-, the same as the sequence found for canine intestinal CCK-58 from this pool of dogs. This is the same sequence others have reported for porcine brain CCK-58 lacking nine amino acid residues (CCK-58 desnonapeptide) except that the porcine peptide had a serine in position 9. The canine CCK amino-terminal sequence differed from the sequence Ala-Gln-Lys-Val-Asn-Ser previously reported for intestinal CCK-58 purified from another pool of dog tissue, but the rest of the residues identified were identical in the two peptides. CCK-58 may be a molecular precursor of the smaller forms of CCK in brain as well as in gut.
Introductory analysis of the GTP-cap phase-change kinetics at the end of a microtubule.
Authors: Hill T L
Published: May 2006

An introductory analysis is provided for the two-phase macroscopic kinetic model of the end of a microtubule. Some general relations are derived for one end of a very long microtubule in solution but the main results refer to the steady-state properties of microtubules grown on nucleated sites, as in the experiments of Mitchison and Kirschner [Mitchison, T. & Kirschner, M. W. (1984) Nature (London), in press]. The two-phase model makes it possible to understand qualitatively how long microtubules can grow well below the critical concentration and also how grown microtubules can rapidly disappear from a nucleated site by shortening following a phase change.
A protein kinase activity tightly associated with Drosophila type II DNA topoisomerase.
Authors: Sander M , Nolan J M , Hsieh T
Published: May 2006

A protein kinase activity has been identified that is tightly associated with the purified Drosophila type II DNA topoisomerase. The kinase and topoisomerase activities are not separated when the enzyme is subjected to analytical chromatography (phosphocellulose, single-strand DNA agarose, and Sephacryl S-300) and analytical glycerol gradient sedimentation. These two activities are also inactivated to the same extent by either heat or N-ethylmaleimide treatment. The evidence, however, does not rule out the possibility that the kinase activity resides in a polypeptide other than the topoisomerase polypeptide. The topoisomerase-associated protein kinase activity is not stimulated by Ca2+ or cyclic nucleotides. It shows a broad substrate range, including the DNA topoisomerase itself, casein, phosvitin, and histones. Phosphoamino acid analysis identified phosphoserine and phosphothreonine in polypeptides modified by the topoisomerase-associated protein kinase. No similar activity has been identified previously in Drosophila melanogaster.
Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome.
Authors: Fleischmann G , Pflugfelder G , Steiner E K , Javaherian K , Howard G C , Wang J C , Elgin S C
Published: May 2006

The distribution of DNA topoisomerase I within Drosophila polytene chromosomes was observed by immunofluorescent staining with affinity-purified antibodies. The enzyme is preferentially associated with active loci, as shown by prominent staining of puffs. The heat shock loci 87A-87C are stained after, but not before, heat shock induction. A detailed comparison of the distribution of topoisomerase I with that of RNA polymerase II reveals a similar, although not identical, pattern of association. Topoisomerase I is also found in association with the nucleolus, the site of transcription by RNA polymerase I.
Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.
Authors: Hershko A , Heller H , Eytan E , Kaklij G , Rose I A
Published: May 2006

Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation.
Construction of a retrovirus capable of transducing and expressing genes in multipotential embryonic cells.
Authors: Rubenstein J L , Nicolas J F , Jacob F
Published: May 2006

Retroviral gene expression is inhibited in embryonal carcinoma (EC) cells. We have constructed a recombinant retroviral vector that is capable of expressing the neomycin-resistance (neo) gene in EC cells. The critical modification that permits expression of the neo gene is the insertion of a composite simian virus 40 early gene-herpes simplex virus type 1 thymidine kinase gene (SVtk) promoter 3' to the viral first intron and 5' to the neo gene. When the SVtk promoter is deleted, the recombinant retrovirus is either unable or extremely inefficient at expressing the neo gene in EC cells.
Condensation of nucleic acids by intercalating aromatic cations.
Authors: Kapuscinski J , Darzynkiewicz Z
Published: May 2006

Certain intercalating aromatic cations, such as the fluorochrome acridine orange or the antitumor drug Mitoxantrone, induce condensation of nucleic acids in solutions. The appearance of the condensed form during titration of nucleic acids with these intercalating ligands can be quantitatively monitored by light scatter measurements. The resulting highly reproducible light scatter transition curves are typical of the cooperative processes, and the transitions occur at different critical concentrations of the ligands depending upon both the ligand itself and the primary structure (base and sugar composition) and the secondary structure (single- or double-stranded) of the nucleic acids. The mechanism of condensation of nucleic acids by intercalating cationic ligands is discussed in light of the model of interactions occurring between certain intercalators and single-stranded nucleic acids and compared with the condensation induced by polyvalent "simple" cations such as Co3+ or spermine4+. The described phenomenon can have an application in analytical and preparative biochemistry for characterization of the primary and secondary structure of nucleic acids and for separation of the compounds. The possibility that the condensation plays a role in mutagenic and pharmacological effects of aromatic cations is considered.
Nucleotide sequence of the insertion sequence found in the T-DNA region of mutant Ti plasmid pTiA66 and distribution of its homologues in octopine Ti plasmid.
Authors: Machida Y , Sakurai M , Kiyokawa S , Ubasawa A , Suzuki Y , Ikeda J E
Published: May 2006

The octopine tumor-inducing (Ti) plasmid pTiA66 has an insertion mutation in its T region (the DNA region incorporated into the plant genome) that results in the slow growth of crown gall tumors. These tumors exhibit hormonal autonomy different from that of the crown gall tumors caused by wild-type Ti plasmids. In the present study, the nucleotide sequences of both the DNA segment inserted into pTiA66 and its target site have been determined. The inserted segment is 2548 base pairs long and has 20-base-pair terminal inverted repeats. An 8-base-pair sequence at the target site is duplicated at both integration junctions. These structural features of the insert suggest that it is a bacterial insertion sequence (IS) element, which we have named IS66. Blot-hybridization analyses using IS66 probes revealed that genomes of octopine Ti plasmids contain at least three sequences homologous to IS66: two homologues are located in the virulence region and one is located between the left-hand (TL-DNA) and right-hand (TR-DNA) portions of T-DNA. The chromosome of Agrobacterium tumefaciens A66 also contains two sequences highly homologous to IS66. These results suggest that the mutant pTiA66 plasmid was generated by translocation of one of the sequences showing homology with IS66 into the T region. The fact that a sequence homologous to IS66 is present between TL-DNA and TR-DNA also suggests that the octopine T region was split into two portions, TL-DNA and TR-DNA, by translocation of IS66 or its relatives. Thus, IS66 may cause genetic and structural variations of the T region and the vir region of the octopine Ti plasmids.
Enhanced recombination between lambda plac5 and F42lac: identification of cis- and trans-acting factors.
Authors: Seifert H S , Porter R D
Published: May 2006

Enhanced transductional recombination between specialized transducing phage lambda plac5 and plasmid F42lac depends on cis- and trans-acting factors. By constructing a series of recombinant molecules, the cis-acting site required for enhanced recombination has been identified as oriT, the origin of conjugational transfer of the F sex factor of Escherichia coli. The trans-acting factors are located in the promoter-proximal and the promoter-distal regions of the traY-to-traZ operon.
The protein encoded by the human proto-oncogene c-myc.
Authors: Ramsay G , Evan G I , Bishop J M
Published: May 2006

The proto-oncogene c-myc may play a role in controlling the growth and division of normal cells, and abnormalities of the gene have been implicated in the genesis of a substantial variety of human tumors. To facilitate further study of these issues, we developed antisera that permit the identification and isolation of the protein encoded by the human and other mammalian versions of c-myc. We found that c-myc(human) gives rise to at least two phosphoproteins with apparent molecular weights of 62,000 [pp62c-myc(human), the major product] and 66,000 [pp66c-myc(human), produced in smaller quantities and possibly a modified version of the Mr 62,000 protein]. Both proteins have relatively short half-lives of approximately equal to 30 min. Mouse c-myc encodes similar proteins with molecular weights of 64,000 and 66,000. The use of cells transformed by DNA-mediated gene transfer sustained previous deductions that the entire coding domain of c-myc(human) is contained in the second and third exons of the gene and resolved previous ambiguities by showing that analogous exons specify the entire protein product of c-myc(chicken). Tumor cells containing amplification of c-myc(human) produce relatively large amounts of pp62/pp66c-myc(human). By contrast, translocations of c-myc found in cells derived from Burkitt lymphoma appear merely to sustain expression of c-myc(human) at levels found also in nontumorigenic lymphoblastoid cells, rather than to increase expression of the gene to manifestly abnormal levels.
Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture.
Authors: Allen-Hoffmann B L , Rheinwald J G
Published: May 2006

We have developed a culture system for detecting and isolating rare hypoxanthine phosphoribosyltransferase-deficient mutants of human epidermal keratinocytes. A thioguanine-resistant variant, 3T3M1, of the Swiss mouse fibroblast line 3T3 was used as a feeder layer to support clonal growth of mutant keratinocytes. A near-diploid, epidermal squamous cell carcinoma line, SCC-13Y, was used as a prototype to determine mutagen treatment conditions, plating density, and phenotypic expression time for maximum mutant recovery. To extend this system to normal keratinocytes, we improved the culture conditions by adding insulin, adenine, and Ham's nutrient mixture F-12, which increased colony-forming efficiencies to 30% in early passage and made feasible the detection of rare mutants in normal epidermal keratinocyte populations. We have quantitated mutation in SCC-13Y and three strains of normal human epidermal keratinocytes after exposure to polycyclic aromatic hydrocarbons, which are activated to their mutagenic forms by cellular mixed-function oxidases. 7,12-Dimethylbenz[a]anthracene and benzo[a]pyrene caused almost no cytotoxicity, but induced thioguanine-resistant mutants at frequencies as much as 50-fold higher than the spontaneous frequency of approximately 10(-6). The mutants were aminopterin-sensitive and possessed no measurable hypoxanthine phosphoribosyltransferase activity; their behavior was indistinguishable from that of keratinocytes cultured from individuals with Lesch-Nyhan syndrome. This mutagenesis assay system should also be applicable to other feeder layer-dependent human epithelial cell types, such as urothelial, mammary, and tracheal epithelial cells.
Upstream activation sites of the CYC1 gene of Saccharomyces cerevisiae are active when inverted but not when placed downstream of the "TATA box".
Authors: Guarente L , Hoar E
Published: May 2006

The ability of the upstream activation sites (UASs) of the yeast CYC1 gene to function when inverted or when positioned downstream of the "TATA box" is investigated. Inversion of a 130-base-pair DNA fragment bearing the UASs leaves the activity of the sites almost completely intact. In contrast, positioning the sites downstream of the TATA box or in the intron of a CYC1-ribosomal protein 51-lacZ tribrid gene almost totally abolishes their activity. In the latter construct, the separation between the UASs and TATA box is roughly equivalent to that between the elements in the intact CYC1 promoter region. The UASs are shown not to interrupt transcription of splicing in this construct since a GAL10 UAS positioned upstream of the TATA box gives rise to galactose-inducible expression of the tribrid gene. The inability of the UASs to function in the intron is partly due to sequences between the intron and the TATA box that block the activation signal. However, a large component of the inactivity of the sites in the intron appears to be their downstream location. This result is discussed in light of possible mechanisms of upstream activation in yeast.
Primary structure of an amyloid prealbumin and its plasma precursor in a heredofamilial polyneuropathy of Swedish origin.
Authors: Dwulet F E , Benson M D
Published: May 2006

Prealbumin from an individual with heredofamilial amyloid polyneuropathy of Swedish origin was isolated from plasma by using a three-step procedure involving ion exchange, Affi-gel Blue affinity chromatography, and gel filtration. This prealbumin and its associated amyloid fibril subunit protein were digested with trypsin and the resulting peptides were separated by high performance liquid chromatography. Comparison with normal prealbumin peptides showed that an amino acid substitution of a methionine for a valine had occurred at position 30. In the plasma prealbumin, the abnormal residue accounted for 1/3rd of the material while in the amyloid fibrils it accounted for 2/3rds. From this sequence information and the known three-dimensional structure of the prealbumin molecule, a mechanism for the amyloid formation is proposed. It involves formation of the amyloid fibrils by addition of prealbumin dimers or tetramers to the aggregate. Each dimer must contain at least one variant peptide chain while the tetramer must contain at least two abnormal chains. Either of these models can account for the observed amount of normal prealbumin in amyloid fibrils. No proteolytic processing of this molecule is required because the entire undegraded prealbumin molecule is found in the fibrils.
Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin.
Authors: Deschenes R J , Lorenz L J , Haun R S , Roos B A , Collier K J , Dixon J E
Published: May 2006

Poly(A) RNA was isolated from a rat medullary thyroid carcinoma that exhibited high levels of immunoreactive cholecystokinin (CCK). Double-stranded cDNA was synthesized from the poly(A) RNA and inserted into the Pst I site of pBR322. Bacterial colonies containing CCK cDNA were identified using the hybridization probe d(T-C-C-A-T-C-C-A-N-C-C-C-A-T-G-T-A-G-T-C). The sequence of the probe was deduced from the known amino acid sequence of porcine CCK-8, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2. The nucleotide sequence of the cDNA complementary to the mRNA of rat preprocholecystokinin was determined. The cDNA contains 33 nucleotides in the 5'-noncoding region, 199 nucleotides in the 3'-noncoding region, and 345 nucleotides coding for a precursor to CCK, which is 115 amino acids (Mr, 12,826). Examination of the rat CCK gene revealed a suggested transcriptional control sequence analogous to the "TATA" sequence located 33 nucleotides upstream from a proposed transcriptional start site. The amino acid sequence of CCK-39 is flanked by both amino-terminal and carboxyl-terminal extensions. Analysis of CCK mRNA showed that it is approximately equal to 750 nucleotides long. CCK mRNA of the rat brain and intestine appeared to be identical in size to the CCK mRNA of the carcinoma.
Functional T cells in athymic nude mice.
Authors: Ikehara S , Pahwa R N , Fernandes G , Hansen C T , Good R A
Published: May 2006

After passage of spleen cells from nu/nu mice over a nylon wool column, concanavalin A-responsive cells can be detected in the presence of 2-mercaptoethanol, and specific cytotoxic T lymphocytes can be generated without exposure to interleukin 2 (IL-2). The spleen cells of the nu/nu mice born of nu/nu parents and nursed by nu/nu mothers had significantly fewer Thy-1+ T cells and a lesser capacity to generate cytotoxic T lymphocytes than did the conventionally bred nu/nu mice. Nonetheless, such cells were clearly present. IL-2 may act to cause these post-thymic T cells to proliferate. Therefore, it seems inappropriate to consider IL-2 as an inducer of the differentiation of T cells in the absence of thymic influence on the basis of the capacity of IL-2 to induce the appearance of a T-lymphocyte population in nu/nu mice.
Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action.
Authors: D'Ercole A J , Stiles A D , Underwood L E
Published: May 2006

We have validated a method for extracting and measuring the tissue content of somatomedin C (Sm-C)/insulin-like growth factor I (IGF-I), a growth-hormone-dependent, growth-promoting peptide. The Sm-C content of tissue extracts was strongly growth-hormone dependent because most of the tissues studied from hypophysectomized rats contained significantly less Sm-C than normal tissues. The intraperitoneal administration of ovine growth hormone (oGH) to hypophysectomized rats caused tissue extractable Sm-C to increase in kidney, liver, lung, heart, and testes. Tissue Sm-C responses to oGH were maximal after 12 hr, 6 hr before the maximal increment in serum. In liver and lung, the tissue Sm-C response to various doses of oGH fit linear regression models, and the doses of oGH needed to increase the Sm-C are in the range of those required to increase protein synthesis. Although these results do not exclude the possibility that the somatomedins act by hormone-like endocrine mechanisms, they add support to the concept that these peptides act through autocrine or paracrine mechanisms, being produced at multiple sites and acting at or near their sites of production.
Evaluation of critical groups required for the binding of heparin to antithrombin.
Authors: Atha D H , Stephens A W , Rosenberg R D
Published: May 2006

We have examined the quantitative importance of various monosaccharide residues of an octasaccharide domain of heparin that are responsible for the binding of this oligosaccharide to antithrombin. Different fragments of the octasaccharide were prepared by enzymatic digestion and the avidities of these oligosaccharides for antithrombin were determined by equilibrium dialysis. The data show that the non-reducing-end and the reducing-end tetrasaccharides contribute equally to the binding energy of the octasaccharide. The O6-sulfate group of the N-acetyl glucosamine moiety within the nonreducing-end tetrasaccharide is responsible for approximately equal to 45% of the binding energy of the octasaccharide. Neither the two non-sulfated uronic acid groups that flank this residue nor the N-sulfated glucosamine residue on the reducing end of this tetrasaccharide sequence that bears the unique O3-sulfate substituent contribute significantly to the binding energy of the octasaccharide. We suggest that the lack of sulfation of the two uronic acid moieties within the nonreducing-end tetrasaccharide may be required to permit the N-acetyl glucosamine O6-sulfate group to interact with a specific region on the antithrombin molecule. However, we cannot exclude the possibility that the O3-sulfate group plays an important role in orienting this O6-sulfate group within the nonreducing-end tetrasaccharide.
RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro.
Authors: Ogawa T , Pickett G G , Kogoma T , Kornberg A
Published: May 2006

Escherichia coli rnh mutants defective in RNase H activity display the features of previously described sdrA (stable DNA replication) and dasF (dnaA suppressor) mutants: (i) sustained DNA replication in the absence of protein synthesis, (ii) lack of requirement for dnaA protein and the origin of replication (oriC), and (iii) sensitivity of growth to a rich medium. Both the sdrA mutants (selected for continued DNA replication in the absence of protein synthesis) and the dasF mutants (selected as dnaA suppressors) are defective in RNase H activity, measured in vitro. Furthermore, a 760-base-pair fragment containing the rnh+ structural gene complements the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene. One function of RNase H in vivo is in the initiation of a cycle of DNA replication at oriC dependent on dnaA+. In keeping with these results, RNase H contributes to the specificity of dnaA protein-dependent replication initiated at oriC in a partially purified enzyme system.
Amino acid sequence homology among the major outer membrane proteins of Escherichia coli.
Authors: Nikaido H , Wu H C
Published: May 2006

Analysis of amino acid sequences reported for the major outer membrane proteins of Escherichia coli, including the porins (OmpF, OmpC, and PhoE), the phage lambda receptor (LamB), and another protein (OmpA), revealed several regions of local homology that is statistically significant. The implications of this observation are discussed in relation to the evolutionary origins of these proteins, as well as to the mechanism of export of these proteins to the outer membrane.
beta-Thalassemia in American Blacks: novel mutations in the "TATA" box and an acceptor splice site.
Authors: Antonarakis S E , Irkin S H , Cheng T C , Scott A F , Sexton J P , Trusko S P , Charache S , Kazazian H H
Published: May 2006

beta-Thalassemia genes, although often mild in their effects, are common among American Blacks. We have begun a systematic molecular analysis of beta-thalassemia mutations in this group. DNA polymorphisms in the beta-globin gene cluster were examined among 22 beta-thalassemia chromosomes. Six different haplotypes were observed. beta-globin genes of two of these were cloned, and their phenotypes were examined both in heterologous cells upon transient expression and in vivo. The gene found in the most common haplotype (9 of 22 chromosomes) contained a single base substitution (A----G) at position -29 within the highly conserved proximal promoter element (the "TATA" box). This mutant gene directed beta-globin RNA at 25% of normal levels both in heterologous cells and in vivo. It was associated with a mild beta +-thalassemia phenotype. A different gene, isolated from an apparently rare haplotype (1 of 22 chromosomes), had a single base substitution (A----G) within the acceptor splice site of the second intervening sequence. This mutation abolished normal RNA splicing so that the only RNA made from the gene in vitro was an alternatively spliced RNA, which could not encode beta-globin. The mild deficit in beta-globin production attributable to the -29 A----G mutant allele most likely accounts for the frequently mild nature of beta-thalassemia among American Blacks.
Fibroblasts from patients with hereditary cutaneous malignant melanoma are abnormally sensitive to the mutagenic effect of simulated sunlight and 4-nitroquinoline 1-oxide.
Authors: Howell J N , Greene M H , Corner R C , Maher V M , McCormick J J
Published: May 2006

Because of a possible etiologic link between mutations and carcinogenesis, we compared fibroblasts derived from skin biopsies of several patients with hereditary cutaneous malignant melanoma and the dysplastic nevus syndrome for sensitivity to the mutagenic and/or cytotoxic effect of broad-spectrum simulated sunlight and of a UV mimetic carcinogen, 4-nitroquinoline 1-oxide (4NQO). The genetic marker was resistance to 6-thioguanine; loss of colony-forming ability was the assay for cytotoxicity. All five strains tested were more sensitive than normal to the killing effect of 4NQO (slopes of survival curves were 2- to 3-fold steeper), but only one strain was hypersensitive to killing by Sun Lamp radiation. Two strains were tested for mutagenicity. The response of each to the mutagenic action of these agents corresponded to its response to cell killing. Both strains were hypermutable after exposure to 4NQO, but only one showed a higher than normal frequency of mutants induced by simulated sunlight. The finding that nonmalignant fibroblasts from patients with a hereditary variant of malignant melanoma are abnormally susceptible to carcinogen-induced mutations suggests that hypersensitivity to mutagens contributes to risk of melanoma in patients. It also supports the somatic cell mutation hypothesis for the origin of cancer.
Regioselective arylation of ribose in adenosine and guanosine with the antitumor drug N2-methyl-9-hydroxyellipticinium acetate.
Authors: Bernadou J , Meunier B , Meunier G , Auclair C , Paoletti C
Published: May 2006

The transformation of the antitumor drug N2-methyl-9-hydroxy-ellipticinium by a peroxidase-hydrogen peroxide system, which has been shown to occur in vivo, leads to an electrophilic quinone-imine derivative. This unstable molecule arylates in vitro purine nucleosides and nucleotides, leading to regioselective adducts substituted only at the 2'-O position of the ribose, as shown by mass spectrometry and NMR. It is likely that an important preliminary step in this reaction is a stacking process between the ellipticinium ion and the purine rings, which might explain this regioselectivity.
The free energy of DNA supercoiling is enthalpy-determined.
Authors: Seidl A , Hinz H J
Published: May 2006

The thermodynamics of superhelix formation was determined by combining superhelix density data with enthalpy values obtained from microcalorimetric measurements of the relaxation of supercoiled ColE1 amp plasmid DNA in the presence of topoisomerase I from Escherichia coli (omega protein). The thermodynamic quantities for superhelix formation at 37 degrees C in 10 mM Tris/2 mM MgCl2/1 mM EDTA pH 8, are: delta G = 921 kJ X (mol of plasmid)-1; delta H 2260 kJ X (mol of plasmid)-1; deltaS = 4.3 kJ X (mol of plasmid X K)-1. These data clearly demonstrate that the unfavorable Gibbs free energy associated with supercoiling of DNA results exclusively from the positive enthalpy involved in formation of superhelical turns. A positive overall entropy change accompanies superhelix formation, which overcompensates the expected decrease of configurational entropy. By neglecting contributions from bending, an estimate of the torsional rigidity C = 1.79 X 10(-19) erg X cm (1 erg = 0.1 microJ) of the supercoiled ColE1 amp plasmid DNA was made on the basis of the enthalpy value. This value is in excellent agreement with values of C derived from subnanosecond time-resolved fluorescence depolarization measurements for pBR322 DNA [Millar, D. P., Robbins, R. J. & Zewai, A.H. (1982) J. Chem. Phys. 76, 2080-2094]. The magnitude of C is larger than for linear DNAs, indicating that supercoiled DNA is more rigid than linear DNA.
9-Deoxy-delta 9, delta 12-13,14-dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma.
Authors: Kikawa Y , Narumiya S , Fukushima M , Wakatsuka H , Hayaishi O
Published: May 2006

Incubation of prostaglandin D2 (PGD2) with human plasma yielded a product that has been identified as 9-deoxy-9,10-didehydro-12,13-didehydro-13,14-dihydro-PGD2 (9-deoxy-delta 9, delta 12-13,14-dihydro-PGD2). The identification was based on mass spectrometry, UV spectrometry, mobilities and retention time on TLC and HPLC, and NMR. The conversion of PGD2 to this product was dependent on the incubation time and the amount of plasma added to a reaction mixture and was abolished by prior boiling. The conversion rate of PGD2 to this metabolite was 0.03 nmol/min per mg of protein of whole plasma at pH 8.0 at 37 degrees C. Similar conversion was also found by incubating PGD2 with human serum albumin added at the concentration found in plasma. These results suggest that the conversion of PGD2 to this product is catalyzed by the enzymatic action of a plasma protein, probably serum albumin. The biological activities of this compound were examined in several systems. It showed negligible activity in inhibition of human platelet aggregation and relaxation of rabbit stomach strip. On the other hand, it exhibited a three times stronger inhibitory activity (IC50, 1.8 microM) than PGD2 (IC50, 5 microM) on the growth of L-1210 cultured cells.