bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism by which caffeine potentiates lethality of nitrogen mustard.
Authors: Lau C C , Pardee A B
Published: May 2006

Caffeine is synergistic with many DNA-damaging agents in increasing lethality to mammalian cells. The mechanism is not well understood. Our results show that caffeine potentiates the lethality of the nitrogen mustard 2-chloro-N-(2-chloroethyl)-N-methylethanamine (HN2) by inducing damaged cells to undergo mitosis before properly repairing lesions in their DNA. Treatment with low doses of HN2 (0.5 microM for 1 hr) caused little lethality in baby hamster kidney cells (90% survival). These cells were arrested in G2 shortly after treatment with HN2 as shown by flow microfluorimetry and autoradiography. After an arrest of 6 hr, HN2-treated cells began to move into mitosis and from then on behaved like normal cells. Repair synthesis was shown to continue during the G2 arrest by using synchronized cells pulse labeled with [3H]thymidine after HN2 treatment and autoradiography. Caffeine (2mM) increased the lethality of HN2 by 5- to 10-fold. It prevented the G2 arrest. Caffeine did not prevent these HN2-treated cells from entering or completing S phase but rather allowed them to divide without finishing the repair processes and as a consequence caused nuclear fragmentation after mitosis. Caffeine-induced nuclear fragmentation and enhanced lethality were proportional, as shown with dose--response curves and time dependence. In addition, both lethality and nuclear fragmentation were abolished by low doses of cycloheximide, an inhibitor of protein synthesis.
Construction and characterization of genomic libraries from specific human chromosomes.
Authors: Krumlauf R , Jeanpierre M , Young B D
Published: May 2006

Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.
Human gamma-trace, a basic microprotein: amino acid sequence and presence in the adenohypophysis.
Authors: Grubb A , Löfberg H
Published: May 2006

The amino acid sequence of human gamma-trace, a basic microprotein without known function, was determined by automated Edman degradations of the carboxymethylated polypeptide chain and of fragments obtained by cyanogen bromide treatment and tryptic digestion after blocking of lysine residues. The single polypeptide chain contained 120 residues, and the calculated Mr was 13,260. A proline residue at position 3 was partly hydroxylated. The presence of gamma-trace in a significant proportion of the cells in the anterior lobe of simian and human pituitary glands was demonstrated by immunohistochemical procedures with a rabbit antiserum against human gamma-trace. The tissue localization and amino acid sequence of gamma-trace indicated that this protein is connected with the peptidergic gastroenteropancreatic neuroendocrine system.
Immunohistochemical distribution of alpha-neo-endorphin/dynorphin neuronal systems in rat brain: evidence for colocalization.
Authors: Weber E , Roth K A , Barchas J D
Published: May 2006

alpha-Neo-endorphin and dynorphin immunoreactivities in rat brain were visualized by double antibody immunofluorescence of frozen sections. Antibodies were used that were specific for their respective antigens. The pattern of neuronal immunostaining produced by alpha-neo-endorphin and dynorphin antisera in adjacent serial sections was completely superimposible. No areas were found in which alpha-neo-endorphin or dynorphin immunoreactivities existed alone. The following brain regions contained alpha-neo-endorphin/dynorphin-immunoreactive fibers and terminals: the median forebrain bundle, the internal capsule, the substantia nigra, the hypothalamus, the nucleus accumbens, the hippocampus, and the medulla oblongata. A few fibers were seen in the cerebral cortex and in the corpus striatum. In many regions, this neuronal fiber system seems to overlap the neuronal system previously described to contain [Met]-/[Leu]enkephalin-immunoreactive material. In brains from colchicine-treated animals, numerous alpha-neo-endorphin/dynorphin-immunoreactive neuronal cell bodies were seen in the supraoptic, retrochiasmatic supraoptic, paraventricular, and magnocellular accessory nuclei of the hypothalamus. It is concluded that alpha-neo-endorphin-like and dynorphin-like immunoreactivities are part of the same neuronal system.
Secretion of catecholamines from adrenal gland by a single electrical shock: electronic depolarization of medullary cell membrane.
Authors: Wakade A R , Wakade T D
Published: May 2006

Transmural stimulation of the isolated adrenal gland of the rat and guinea pig results in secretion of catecholamine. The secretion is due to activation of cholinergic receptors of the adrenal medulla by acetylcholine released from splanchnic nerve terminals after transmural stimulation. Our aim was to see whether the same experimental technique could be used to directly excite the adrenal medullary cell membrane by electrical stimulation and whether such stimulation would result in secretion of catecholamines. We demonstrate here that a single electrical shock to the perfused adrenal gland of the rat results in massive secretion of epinephrine and norepinephrine. The secretion is directly related to the strength and duration of the applied stimulus over a wide range. Catecholamine secretion is unaffected by tetrodotoxin or hexamethonium/atropine but is abolished by Ca2+ lack or 3 mM Mn2+. We suggest that the adrenal medullary membrane undergoes nonpropagated electrotonic depolarization on electric stimulation and thereby voltage-dependent Ca2+ channels are opened to initiate secretion.
Organization of Achlya mtDNA: a population with two orientations and a large inverted repeat containing the rRNA genes.
Authors: Hudspeth M E , Shumard D S , Bradford C J , Grossman L I
Published: May 2006

We have investigated mtDNA organization in the oömycetous water mold; Achlya, and report here that this primitive organism contains a circular mitochondrial genome of 49.8 kilobase pairs. Extensive restriction endonuclease analysis indicates that a significant portion of the genome is present as an inverted repeat. Of 52 restriction sites for 14 enzymes thus far mapped, 28 sites cluster in two 9.6-kilobase-pair regions; within these regions, the sequence of sites is inverted but the spacing between analogous sites is identical. The repeat arms have a maximum length of 12.1 kilobase pairs and are separated by 4.6-8.4 and 21.0-22.3 kilobase pairs of unique sequences. Transfer hybridization experiments show that genes for both the large and the small rRNAs are contained within each repeat. Restriction endonuclease analysis shows that the unique regions between the inverted repeats are present in both possible orientations with respect to each other and in approximately equal proportions. These orientational, or "flip-flop," isomers of the unique regions are postulated to occur by intramolecular homologous recombination between the repeated regions.
Redistribution of fodrin (a component of the cortical cytoplasm) accompanying capping of cell surface molecules.
Authors: Levine J , Willard M
Published: May 2006

Fodrin, a protein composed of two polypeptides with molecular weights of 250,000 and 240,000, is concentrated in the cortical cytoplasm of neurons, and moves down the axons by the process of axonal transport. We have used immunofluorescence techniques to determine whether fodrin antigens also move in non-neuronal cells when cell surface ligands are induced to redistribute by crosslinking them. A redistribution of fodrin antigens occurred in the following instances: (i) when 3T3 cells were incubated with concanavalin A and anti-concanavalin A, surface concanavalin A receptors formed aggregates and fodrin antigens formed corresponding intracellular aggregates; (ii) when B lymphocytes were incubated with anti-Ig, the surface Ig formed caps and fodrin antigens formed intracellular subcaps; (iii) when T lymphocytes were treated with anti-H-2 followed by a secondary antibody, the H-2 antigen formed caps and fodrin formed corresponding subcaps. These observations show that fodrin antigens can move within non-neuronal cells, as well as in axons, and that their organization can be regulated by interaction between surface proteins and environmental stimuli. They also raise the possibility that fodrin, together with other proteins that form subcaps in lymphocytes (e.g., actin, myosin, and alpha-actinin) is a component of the cellular machinery responsible for the capping process. We consider whether the similarities between the movements of fodrin in lymphocyte capping and axonal transport may indicate that certain aspects of these two processes are related.
Apolipoprotein E synthesis in human kidney, adrenal gland, and liver.
Authors: Blue M L , Williams D L , Zucker S , Khan S A , Blum C B
Published: May 2006

Human tissues were incubated in vitro with radiolabeled amino acids to determine whether plasma apolipoproteins are synthesized in human kidney. Subsequently, tissue extracts were screened with antisera directed against apolipoprotein E (apo E), apolipoprotein B (apo B), apolipoprotein AI (apo AI), and bulk apolipoproteins of high density lipoprotein (HDL). Newly synthesized apo E, but not apo AI or apo B, was identified in kidney and adrenal cortex. Estimates of relative rates of apo E synthesis in vitro suggest that a substantial portion of adrenal and kidney protein synthesis is committed to apo E synthesis. The relative rate of apo E synthesis was 4-6 times greater in kidney cortex than in kidney medulla. Analysis of immunoreactive apo E showed that kidney and adrenal apo E species have the same electrophoretic mobility in NaDodSO4/polyacrylamide gels as does plasma apo E. Further characterization by high resolution two-dimensional gel analysis indicated that the isoforms of newly synthesized kidney and adrenal apo E correspond to specific isoforms of plasma apo E. These findings suggest that apolipoproteins arising from peripheral tissues may play an important role in lipid transport and metabolism.
Identification of the leukemogenic protein of avian myeloblastosis virus and of its normal cellular homologue.
Authors: Boyle W J , Lipsick J S , Reddy E P , Baluda M A
Published: May 2006

The genome of the replication-defective avian myeloblastosis virus (AMV) contains an inserted cellular sequence (amv) that is part of the oncogene responsible for acute myeloblastic leukemia in chickens infected with AMV. Three antisera raised against distinct synthetic peptides predicted from the long open reading frame of amv specifically precipitated the same 48-kilodalton protein (p48amv) from leukemic myeloblasts but not from normal hematopoietic tissue, fibroblasts, or from fibroblasts infected with the AMV helper virus, MAV-1 (myeloblastosis-associated virus type 1). p48amv is not glycosylated or phosphorylated and does not appear to act as a protein kinase in vitro. The same three antisera that recognized p48amv also specifically precipitated a common 110-kilodalton protein from normal uninfected hematopoietic tissue. This normal cellular homologue of the AMV leukemogenic protein, p110proto-amv, was not present in normal fibroblasts, MAV-1 infected fibroblasts, or, interestingly, in some leukemic myeloblasts. We conclude that p48amv is the leukemogenic product of an altered, transduced, partial protooncogene. Short helper-virus sequences provide its carboxyl terminus and also may provide the amino terminus.
Shape determinations of ribosomal proteins in situ.
Authors: Nierhaus K H , Lietzke R , May R P , Nowotny V , Schulze H , Simpson K , Wurmbach P , Stuhrmann H B
Published: May 2006

Ribosomes are heterogeneous for neutrons because RNA and proteins have different neutron-scattering-length densities. This heterogeneity is an obstacle to the shape determination of single ribosomal components within the ribosome. Therefore, we homogenized (matched) the neutron-scattering-length densities of RNA and proteins. 23S and 5S RNA from the large ribosomal subunit were isolated from cells grown in a medium containing 76% 2H2O. The total protein fraction of the large ribosomal subunit was isolated from cells grown in a medium containing 84% 2H2O. When these constituents were used for total reconstitution of 50S subunits, neutron scattering measurements of the reconstituted particles revealed excellent matching near 100% 2H2O. A three-step reconstitution procedure was developed that allowed the reconstitution of 50S subunits from deuterated RNA, deuterated total (i.e., unfractionated) proteins, and single protonated proteins. The reconstituted particles contain one protonated protein or two in a matched ribosomal matrix and were used for shape determination or distance measurement of mass centers of gravity, respectively. The signal/noise ratio is high enough to allow measurement in solutions containing nearly 100% 2H2O at concentrations of only 300-500 A260 nm units/ml. Our experiments have proved the feasibility of our biochemical strategy. The shape determinations of ribosomal proteins in situ gave radii of gyration for L1, L3, L4, and L23 of 26 +/- 2, 22 +/- 2, 20 +/- 2, and 13 +/- 2 A, respectively.
High mutation frequency in DNA transfected into mammalian cells.
Authors: Calos M P , Lebkowski J S , Botchan M R
Published: May 2006

The lacI gene of Escherichia coli was used to score mutation in mammalian cells of simian virus 40-based recombinant DNA vectors that provide for replication and selection in both bacterial and mammalian cells. Plasmid DNA was introduced into COS7 simian cells by DEAE-dextran transfection, allowed to replicate in the mammalian cells, and then returned to E. coli for analysis. Mutants in lacI were observed at frequencies of one to several percent, compared with a spontaneous mutation rate in E. coli of less than 10(-5). The lesions include a large number of base substitutions, in addition to deletions, duplications, and more complex rearrangements, including insertion into the plasmid of sequences originating in the host genome. We discuss possible sources of the high mutation frequency and its implications for experiments involving DNA transfer.
Molecular topography of the neural cell adhesion molecule N-CAM: surface orientation and location of sialic acid-rich and binding regions.
Authors: Cunningham B A , Hoffman S , Rutishauser U , Hemperly J J , Edelman G M
Published: May 2006

Chemical analyses and binding studies have been correlated to clarify the relationship of structure to function in the neural cell adhesion molecule (N-CAM) from embryonic chicken brain. N-CAM isolated from the cell surface appears to include two closely related polypeptide chains. Treatment with neuraminidase of such preparations of N-CAM bound by antibodies on solid supports yielded components of Mr 140,000 and 170,000. These components each had the same amino-terminal sequence as N-CAM and gave nearly identical profiles on peptide maps. Immunoprecipitation of N-CAM from 9-day brain cells treated with tunicamycin yielded corresponding components of Mr 130,000 and 160,000, suggesting that the differences between these two components of N-CAM are in the polypeptide rather than the carbohydrate portions of the molecules. N-CAM appears to be oriented with the amino terminus extending away from the cell surface and with the bulk of the sialic acid near the middle of the peptide chain. As shown previously, incubation of N-CAM at 37 degrees C generates a fragment (Fr1) of Mr 65,000 that lacks most of the sialic acid. Treatment of membranes with Staphylococcus aureus V-8 protease released a fragment (Fr2) of N-CAM that contained most of the sialic acid; this fragment had an Mr of 108,000 after neuraminidase treatment. Both of these fragments contain the amino-terminal portion of the polypeptide chain. At least a portion of the N-CAM binding site was found to be located in the amino-terminal region of the peptide chain. Most or all of the sialic acid was not directly involved in binding, although it can influence binding, as indicated by the finding that neuraminidase-treated N-CAM (desialylated-N-CAM) bound to cells to a greater extent than untreated N-CAM. The Fr1 and the Fr2 fragments in solution did not bind to cells but were as effective as N-CAM and desialylated-N-CAM as competitors for N-CAM binding to cells. When fixed covalently to beads, N-CAM, desialylated-N-CAM, and the Fr1 and Fr2 fragments bound specifically to cells. In contrast, the N-CAM autolysis products released along with Fr1 neither bound to cells nor competed for N-CAM binding. In addition to suggesting a location for the N-CAM binding region, the accumulated results raise the possibility that valence may play a key role in N-CAM binding.
Inhibition of eukaryotic protein chain initiation by vanadate.
Authors: Ranu R S
Published: May 2006

Vanadate inhibits protein chain initiation in rabbit reticulocyte lysates. The evidence that supports this conclusion is as follows: (i) the biphasic kinetics of inhibition in which protein synthesis is maintained at the control rate for 1-2 min is followed by an abrupt decline in the rate of synthesis; (ii) inhibition is associated with a marked disaggregation of polyribosomes and a concomitant increase in 80S ribosomes; and (iii) vanadate concentrations that inhibit protein chain initiation do not inhibit polypeptide chain elongation or the aminoacylation of tRNA. In partial reactions of protein chain initiation, vanadate concentrations that inhibit protein synthesis have no detectable effect on the formation of eukaryotic initiation factor eIF-2-promoted ternary complex with Met-tRNAf and GTP and on the assembly of 40S ribosomal subunit-Met-tRNAf complexes. On the addition of mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are transformed into 80S ribosome-mRNA-Met-tRNAf complexes, termed 80S initiation complexes. In vanadate-treated samples, however, these 80S initiation complexes are defective and unable to proceed beyond this step.
Phosphorylation activates the insulin receptor tyrosine protein kinase.
Authors: Rosen O M , Herrera R , Olowe Y , Petruzzelli L M , Cobb M H
Published: May 2006

Preparations of insulin receptor from cultured 3T3-L1 adipocytes and human placenta previously was found to catalyze the phosphorylation of the 90,000-dalton component of the insulin receptor on tyrosine residues. This insulin-dependent phosphorylation has now been shown to coincide with the generation of an activated, insulin-independent, receptor protein kinase. Activation is dependent upon ATP, divalent cations (Mg2+ and Mn2+), and insulin (half-maximal activation occurs at 6-8 nM insulin). The time required for activation is consistent with that needed for insulin-dependent self-phosphorylation of the receptor present in eluates from wheat germ lectin-agarose columns and in preparations of affinity-purified placental receptor. Activation proceeds unabated in the presence of soybean trypsin inhibitor at 0.1 mg/ml and the activated, insulin-independent, protein kinase sediments in 5-20% sucrose gradients at the same position as the unmodified receptor. Under steady-state conditions, the phosphorylated receptor binds insulin in the same fashion as the unmodified receptor. It is proposed that the self-phosphorylated form of the receptor is the insulin-activated protein kinase that catalyzes the phosphorylation of exogenous protein and peptide substrates. A corollary of this hypothesis is that enzymatic dephosphorylation may be essential for reversibly terminating the activity of the insulin-receptor protein kinase.
RNAs from two independently isolated defective interfering particles of Sindbis virus contain a cellular tRNA sequence at their 5' ends.
Authors: Monroe S S , Schlesinger S
Published: May 2006

Defective interfering (DI) particles are deletion mutants that interfere specifically with the replication of homologous standard virus. We have determined the 5'-terminal nucleotide sequences of two DI RNA populations by the following methods: (i) cloning of the cDNA from one of the DI RNA populations and sequencing a representative clone, and (ii) using both DI RNA populations as templates for preparing primer-directed cDNA transcripts and sequencing these transcripts. The 5' terminal sequences of the two DI RNA populations were not derived from standard Sindbis viral RNA but were almost identical to those of a cellular tRNAAsp.
Family of middle repetitive DNA sequences in the mouse genome with structural features of solitary retroviral long terminal repeats.
Authors: Wirth T , Glöggler K , Baumruker T , Schmidt M , Horak I
Published: May 2006

Screening of a 129/J mouse genomic library under nonstringent hybridization conditions with a xenotropic virus-like long terminal repeat (LTR) probe revealed a family of sequences resembling insertion elements (IS) with structural features of solitary retroviral LTRs; these are called LTR-IS. They are interspersed among variable flanking regions of mouse DNA and lack any viral structural genes. LTR-IS elements start and end with 11-base-pair inverted repeats and contain signals implicated in RNA polymerase II transcriptional regulation: C-C-A-A-T, T-A-T-A-A-A, and A-A-T-A-A-A. The members of the family are homologous, but not identical, approximately equal to 500-base-pair-long elements with 4-base-pair target-site duplications on both sites of the element. There are 500 LTR-IS per mouse haploid genome.
Translational regulation of a specific gene during oogenesis and embryogenesis of
Drosophila
Authors: Fruscoloni Paolo , Al-Atia Ghada R. , Jacobs-Lorena Marcelo
Published: May 2006

Polysomal and postpolysomal mRNAs were prepared from
Drosophila
egg chambers or embryos of different developmental stages. Cell-free translation of these mRNAs followed by two-dimensional gel electrophoresis of the products indicated the presence of a specific mRNA that appears to be translated (polysome-associated) during oogenesis. This mRNA, designated T1 mRNA, is selectively excluded from polysomes in 3-hr- and 5-hr-old embryos and is again translated in 18-hr-old embryos. A clone containing DNA complementary to T1 mRNA was selected from a library of recombinant DNA prepared from polyadenylylated ovary RNA. This clone was positively identified by hybrid-selected translation followed by two-dimensional gel electrophoresis and autoradiography. T1 mRNA is polyadenylylated and codes for a small, acidic protein. The cloned probe hybridizes to a unique site (2L-39CD) of the polytene chromosomes, very close to the histone genes. The results suggest that this mRNA is under specific translational regulation in contrast to a background of a large number of other abundant mRNAs that are translated at all developmental stages examined.
Monoclonal antibodies against HLA-DR antigens replace T helper cells in activation of B lymphocytes.
Authors: Palacios R , Martinez-Maza O , Guy K
Published: May 2006

In the presence but not in the absence of pokeweed mitogen (PWM), monoclonal antibodies against HLA-DR antigens 147 and 164 helped highly purified B lymphocytes to proliferate and mature to Ig-secreting cells. In contrast, neither anti-DR antibody 231 nor the UCHT1 monoclonal anti-human T cell antibody (both of the same isotype as the 147 and 164 anti-DR antibodies) exhibited any helper activity on B cells. B cells pulsed with PWM and subsequently cultured in the presence of anti-DR antibody 147 or 164 proliferated and secreted Ig, whereas B cells that first were pulsed with antibody 147 or 164 and then incubated with PWM did not. PWM alone did not induce any of these responses on purified B cells. Also, antibodies 147 and 164 significantly increased the number of Ig-secreting cells obtained from the WT51 Epstein-Barr virus-transformed B-cell line. Anti-DR antibody 231 inhibited the helper activity on WT51 cells mediated by anti-DR antibody 164. Finally, anti-DR antibodies 147 and 164 assisted B cells from the spleen of athymic nu/nu mice (which bear I-E-encoded products) to produce IgM antibodies against sheep erythrocytes. However, these antibodies had no effect on mouse B cells which do not express on their surface I-E-encoded Ia antigens. Taken collectively, these findings suggest that the structures detected by anti-DR antibodies 147 and 164 on B lymphocytes function as acceptors/transducers of T-cell-derived helper signals.
High-affinity binding of agonists to beta-adrenergic receptors on intact cells.
Authors: Toews M L , Harden T K , Perkins J P
Published: May 2006

The interactions of agonists and antagonists with beta-adrenergic receptors on intact 1321N1 human astrocytoma and C62B rat glioma cells were studied by using the radioligand (-)-[125I]iodopindolol. Competition binding assays were performed at 37 degrees C under equilibrium conditions and in short-time nonequilibrium assays that approximated initial velocity conditions for binding of the radioligand. The theoretical basis and experimental validation of the initial velocity approach for determining binding affinities of rapidly equilibrating ligands are presented. For the agonists isoproterenol and epinephrine, high binding affinities that approximated their apparent affinities for binding in membranes and for increase of cyclic AMP concentrations in intact cells could be demonstrated only in short-time assays; in contrast, much lower affinities were observed with equilibrium (60-min) assays as reported previously for various cell lines. High-affinity binding of isoproterenol to 1321N1 cells also was observed in equilibrium (6-hr) binding assays carried out on ice. These results indicate that in the native state the intact cell beta-adrenergic receptor has a high binding affinity for agonists and suggest that incubation at 37 degrees C in the presence of an agonist converts the receptors to a form with low affinity for agonists.
Activation of DNA ligase by poly(ADP-ribose) in chromatin.
Authors: Ohashi Y , Ueda K , Kawaichi M , Hayaishi O
Published: May 2006

To elucidate the molecular mechanism by which poly(ADP-ribose) participates in DNA excision repair, we examined the effect of poly(ADP-ribose) on DNA ligase activity in DNA/histone and reconstituted chromatin systems. The ligase activity was markedly inhibited by histones; the inhibition varied depending on histone subfraction and DNA/histone ratio. Poly(ADP-ribose), either exogenous or synthesized in situ by poly(ADP-ribose) synthetase, reversed this inhibition by histone almost completely. This effect was specific for poly(ADP-ribose); polyanions such as mRNA, rRNAs, tRNA, and synthetic poly(A) were less effective or ineffective. The ligase activity with reconstituted chromatin as the substrate was about half of that with free DNA whereas the activities with these two substrates were almost the same in the presence of poly(ADP-ribose) synthesized in situ. The polymers synthesized under these conditions were exclusively bound to the synthetase. Together with our previous finding that the enzyme is the main acceptor of the polymer in DNA-damaged cells, these results suggest that poly(ADP-ribose) in the synthetase-bound form counteracts inhibition by histones and activates DNA ligase to rejoin DNA strands in polynucleosomal structures.
Synthesis of calf prochymosin (prorennin) in Escherichia coli.
Authors: Emtage J S , Angal S , Doel M T , Harris T J , Jenkins B , Lilley G , Lowe P A
Published: May 2006

A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach.
Isolation and induction of erythroleukemic cell lines with properties of erythroid progenitor burst-forming cell (BFU-E) and erythroid precursor cell (CFU-E).
Authors: Shibuya T , Mak T W
Published: May 2006

We isolated erythroleukemic cell lines arrested at different levels of the erythroid differentiation pathway. One cell line (CB5), established from mice infected with the helper-independent Friend murine leukemia virus (F-MuLV), exhibited properties similar to those of the normal erythroid progenitor burst-forming cell (BFU-E). Six erythroleukemic cell lines, which were established from the anemia-inducing Friend virus complex (FV-A)-infected mice, formed erythroid colonies similar to the erythroid colony-forming precursor cell (CFU-E) after induction with dimethyl sulfoxide or erythropoietin. Three lines that were established from the polycythemia-inducing Friend virus complex (FV-P)-infected mice also formed low proportions (2-5%) of CFU-E-like colonies after induction by these same inducers. These data, together with the earlier findings that F-MuLV induces an increase in the levels of BFU-E and that FV-A or FV-P stimulates enhancement of CFU-E early after infection, indicate that the erythroleukemic cell lines isolated late in the diseases are at the same levels of differentiation as the leukemic cells in the corresponding initial stages. These cell lines with properties of BFU-E and CFU-E can be induced to differentiate in culture and should add to our understanding of the nature of erythroid progenitor cells and their early differentiation programs.
Monoclonal antibodies inhibit the adhesion of mouse B 16 melanoma cells in vitro and block lung metastasis in vivo.
Authors: Vollmers H P , Birchmeier W
Published: May 2006

Seven monoclonal antibodies against mouse B 16 melanoma cells (produced in syngeneic C57BL/6 mice) were selected that blocked the adhesion of melanoma cells to tissue culture dishes. These antibodies were found to be directed against antigens on the surface of mouse B 16 melanoma cells but not on normal mouse cells such as 3T3 fibroblasts. Similarly, the antigens could not be detected in normal mouse tissues (e.g., lung, kidney, liver) but were found in lungs colonized by B 16 melanoma cells. Significantly, three of these antibodies virtually abolished lung colonization of highly invasive B 16 sublines injected into the animals' bloodstream. They exerted their effect both when preabsorbed by the melanoma cell in vitro and when delivered to the animals prior to the tumor cells. It is suggested that monoclonal antibodies might be a promising tool for preventing metastasis.
Influence of collagen substrata on glycosaminoglycan production by B16 melanoma cells.
Authors: Luikart S D , Maniglia C A , Sartorelli A C
Published: May 2006

A cloned metastatic murine melanoma cell line exhibited similar growth characteristics when propagated on either type I collagen, type IV collagen, or plastic. However, cells grown on both types of collagen exhibited an altered cellular morphology and on type IV collagen only, an increased substrate adhesiveness, relative to those maintained on a plastic substratum. Incorporation of [3H]glucosamine and [35S]sulfate into glycosaminoglycans (GAGs) of cells grown on collagen substrates was 20% and 40% less, respectively, than cells grown on plastic, whereas degradation of cell-associated [35S]sulfate-labeled GAGs was similar in cells grown on collagen or plastic. Although the composition of GAGs was similar in all cultures, consisting of approximately 60% chondroitin and 40% heparin or heparan sulfate, the degree of sulfation of the heparin or heparan sulfate molecules was markedly decreased in cultures grown on collagen. The results indicate that the composition of the extracellular matrix influences the biological behavior of B16 melanoma cells, in part by altering the amount and nature of the GAG molecules produced.
Characterization of monoclonal antibodies to the sperm-specific lactate dehydrogenase isozyme.
Authors: Goldman-Leikin R E , Goldberg E
Published: May 2006

The isozyme of lactate dehydrogenase (LDH) that is specific to testes, designated LDH-C4, is the predominant LDH isozyme in mammalian spermatozoa. Four high-affinity monoclonal antibodies have been developed to murine LDH-C4. These antibodies were tested for crossreactivity with LDH-C4 from rat, hamster, rabbit, and human by competitive binding radioimmunoassays. Monoclonal antibodies RG-1 and RG-2 are specific for adjacent or partially overlapping epitopes. The other two monoclonal antibodies each recognize separate and distinct determinants. One of these, designated RG-4, recognizes a sequential determinant that is contained in the coenzyme binding loop, residues 101-115 of the C subunit. Furthermore, RG-4 shows reduced binding affinity for rat LDH-C4 which differs in amino acid sequence at residue 108 and 111 in this region of the molecule. RG-4 also has reduced affinity for LDH-C4 of other species, which is consistent with substitutions in the amino acid sequences of the coenzyme binding loop. These differences between C4 of closely related species is in contrast to the high degree of conservation of this sequence in the LDH-A4 and LDH-B4 isozymes. These results provide useful information regarding homologies among species of LDH-C4 as well as the evolution of this isozyme.
cDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1.
Authors: Merlie J P , Sebbane R , Gardner S , Lindstrom J
Published: May 2006

Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.
Aphidicolin inhibits the synthesis and joining of short DNA fragments but not the union of 10-kilobase DNA replication intermediates.
Authors: Lönn U , Lönn S
Published: May 2006

DNA replication intermediates in human melanoma cells have been investigated by using the drug aphidicolin, which inhibits DNA polymerase alpha. In untreated cells, Okazaki fragments and 10-kilobase (kb) DNA intermediates are formed. In aphidicolin-treated cells, the replication fork is stopped and there is no formation of DNA replication intermediates. However, 10-kb DNA intermediates formed before the drug blockade are ligated to high molecular weight DNA whereas already formed Okazaki fragments accumulate in the cell. Moreover, in cells released from aphidicolin inhibition there is preferential labeling of 10-kb DNA compared to Okazaki fragments. The 10-kb DNA and the Okazaki fragments, therefore, respond differently to aphidicolin.
Enhancement of toxicity of antitransferrin receptor antibody-Pseudomonas exotoxin conjugates by adenovirus.
Authors: FitzGerald D J , Trowbridge I S , Pastan I , Willingham M C
Published: May 2006

Cytotoxic conjugates were constructed by chemically coupling Pseudomonas exotoxin to antitransferrin receptor antibodies. Toxicity of these conjugates, due to entry via the transferrin receptor, was enhanced 100- to 300-fold in the presence of adenovirus. By electron microscopy and immunofluorescence it was determined that antitransferrin receptor antibodies and the conjugates derived from them entered cells from coated pits into receptosomes. We believe that the enhanced toxicity resulted when adenovirus and the toxin conjugates were internalized into the same receptosomes. In the process of infection, adenovirus enters cells and brings about a virus-mediated disruption of receptosomes; and this disruption can liberate many more toxin molecules into the cytosol than is possible in the absence of virus.
Stimulation of blowfly feeding behavior by octopaminergic drugs
Authors: Long Thomas F. , Murdock Larry L.
Published: May 2006

Adult blowflies (
Phormia regina
Meigen) injected with the octopaminergic drug demethylchlordimeform (10 μg per fly) exhibited enhanced proboscis extension responses when their tarsae were touched to water or aqueous sucrose. They drank more water than saline-injected control flies did but the quantity imbibed was within the normal fluid intake capacity. They became grossly hyperphagic when offered 1 M sucrose, doubling (and in some cases even tripling) their initial body weights. Three other drugs enhanced tarsal responsiveness and induced hyperphagia: DL-octopamine, clonidine (which is known to stimulate octopaminergic receptors in insects), and pargyline, a monoamine oxidase inhibitor. Yohimbine, an antagonist of one class of octopaminergic receptor in insects, prevented the hyperphagia induced by all four drugs. Dopamine, 5-hydroxytryptamine, and DL-norepinephrine failed to cause hyperphagia. These results suggest that octopaminergic receptors in the nervous system of the blowfly positively modulate feeding and drinking behavior.
Denervated sheath cells secrete a new protein after nerve injury.
Authors: Skene J H , Shooter E M
Published: May 2006

When rat sciatic nerves are crushed, Schwann cells or other supporting cells distal to the injury site begin to synthesize and secrete an acidic 37-kilodalton (kDa) protein. This crush-induced protein accumulates within the nerve sheath and accounts for 2-5% of the total extracellular protein in the distal nerve stump. Synthesis of the 37-kDa protein increased for 2 weeks after nerve crush and declines slowly, beginning 4-6 weeks after the injury. The synthesis of the protein may be regulated by axon-Schwann cell contact. The specific induction of the 37-kDa protein and its accumulation in the extracellular space during nerve regeneration suggest that the protein promotes some aspect of axon growth. Because it is induced slowly after injury, the 37-kDa protein is unlikely to stimulate initial outgrowth of axons; however, it might promote later neuronal responses related to axon growth. The sciatic nerve supporting cells also respond to denervation by reducing the synthesis and release of two proteins of molecular mass 51 and 54 kDa. After crush injury to rat optic nerves, glial cells in the distal optic nerve stump also begin to synthesize and release an acidic 37-kDa protein, although axons of this central nervous system tract do not regenerate. If the 37-kDa protein from peripheral nerves proves to participate in the support of axon regrowth, then the results with rat optic nerve suggest that central nervous system glia initiate at least one part of an appropriate response to nerve injury.
Identification of the molecular defect in factor IX Chapel Hill: substitution of histidine for arginine at position 145.
Authors: Noyes C M , Griffith M J , Roberts H R , Lundblad R L
Published: May 2006

Hemophilia B Chapel Hill is a mild hereditary hemorrhagic disorder in which the factor IX antigen is present in normal amounts but factor IX biological activity is markedly reduced. Previous studies have demonstrated that purified factor IX Chapel Hill has 8% of the activity of normal human factor IX and that the activation of factor IX Chapel Hill is defective in that only one of the two peptide bonds hydrolyzed during activation of normal factor IX is cleaved. The tryptic peptides from normal human factor IX and factor IX Chapel Hill were subjected to analysis by high-performance liquid chromatography. Comparison of the elution profile of the peptides obtained from factor IX Chapel Hill and normal factor IX demonstrated that the tripeptide Leu-Thr-Arg, which is derived from the normal molecule (positions 143-145) immediately amino-terminal from the Arg-Ala peptide bond at 145-146 that is cleaved during the activation of factor IX with factor XIa, was absent in the digest obtained from factor factor IX Chapel Hill. The elongated "activation peptide" from factor factor IX Chapel Hill was obtained by further high-performance liquid chromatographic fractionation and subjected to primary structure analysis. The following sequence, corresponding to positions 143-147, was obtained: Leu-Thr-His-Ala-Glu. Thus, the primary molecular defect in factor factor IX Chapel Hill is the substitution of histidine for arginine at position 145. This substitution precludes cleavage by factor XIa at this peptide bond, and the activation peptide region remains associated with the light chain of factor IXa Chapel Hill.
Left-handed Z-DNA in bands of acid-fixed polytene chromosomes.
Authors: Arndt-Jovin D J , Robert-Nicoud M , Zarling D A , Greider C , Weimer E , Jovin T M
Published: May 2006

Antibodies to DNA in the left-handed (Z) conformation bind to acid-fixed polytene chromosomes of both Chironomus thummi and Drosophila melanogaster, as shown by direct and indirect immunofluorescence. Comparison of the phase-contrast, immunofluorescence, and DNA staining patterns shows a predominant localization of the antibody to the regions of high contrast and DNA density, the bands. The immunofluorescence is completely abolished by competition with polynucleotides in the Z conformation but not by those in the B form. DNase but not RNase treatment eliminates the antibody staining. Actinomycin D inhibits binding, whereas mithramycin has no effect. The highly reproducible immunofluorescence patterns obtained with the anti-Z-DNA antibodies demonstrate variations in fluorescence intensity between particular bands, which can be quantitated by laser scanning and photon counting techniques. The telomeric regions and DNA strands associated with end-to-end chromosome linkage and ectopic pairing are exceptionally bright. At saturation, average values of 1 IgG molecule per 3,000 base pairs and 1 per 15,000 base pairs are found in the intensely and weakly staining regions, respectively. An alternative statement is that the left-handed Z-DNA conformation is present at a frequency of 0.02-0.1%. The measured differences reflect variations in the local density of Z-DNA sites and not in the affinity for the specific antibody, which appears to be relatively constant throughout the chromosomes (Kd approximately equal to 10 nM). These observations taken together with results of biophysical studies on the properties of Z-DNA in solution suggest that regions of DNA in the left-handed conformation could be involved in higher-order structural organization of chromosomes and possibly in modulation of their functional state.
Analyses of bromodeoxyuridine-associated sister chromatid exchanges (SCEs) in Bloom syndrome based on cell fusion: single and twin SCEs in endoreduplication.
Authors: Shiraishi Y , Yosida T H , Sandberg A A
Published: May 2006

When Bloom syndrome (BS) cells labeled with bromodeoxyuridine (BrdUrd) for one round of DNA replication were fused with nonlabeled normal cells, the hybrid cells had a normal level of sister chromatid exchange (SCE) at the first mitosis after fusion. However, when normal cells treated with mitomycin C (MC) were fused with nontreated normal cells, the MC-induced SCE was not affected by fusion with normal cells. Single and twin SCEs were analyzed in the Colcemid-induced endoreduplicated normal and BS lymphoid B cells from diplochromosomes. In normal cells, the same number of SCEs occurs in each of the two cell cycles; the SCE ratio of single (6.30 SCEs per cell) to twin (2.92 SCEs per cell) was 2:1 on the endoreduplicated-cell basis, showing 1:1 on the diploid-cell basis. In BS cells, the SCE ratio of single (144.8 SCEs per cell) to twin (5.9 SCEs per cell) was 25:1 on the endoreduplicated-cell basis and was 12:1 on the diploid-cell basis. These studies strongly suggest that most of the BS SCEs occur during the second cell cycle when BrdUrd-containing DNA is used as template for replication and that the normal level of BS SCE observed at the first mitosis of the hybrid cells is the result of SCE inhibition resulting from the fusion with normal cells.
Comparison of secretory protein and membrane composition of secretory granules isolated from normal and neoplastic pancreatic acinar cells of rats.
Authors: Hansen L J , Reddy M K , Reddy J K
Published: May 2006

The diversity of cytodifferentiation in a transplantable rat pancreatic acinar carcinoma provides a biological model system for the study of regulatory molecular events that differ from those in normal acinar cells. Secretory (zymogen) granule proteins and granule membranes of neoplastic and normal pancreatic acinar cells were compared to determine the differences in gene expression between apparently well-differentiated secretory granule-containing neoplastic cells and normal cells. Nineteen proteins observed in two-dimensional polyacrylamide gels of normal secretory granule extracts were seen also in tumor granule extract profiles, with reduced but detectable amounts of lipase and four basic proteins. In addition, tumor granule extracts contained a new protein of Mr 24,000, designated p24, which was not detectable in normal extracts. Neoplastic granule membranes, while having phospholipid composition similar to that of normal membranes, lacked or contained a greatly reduced amount of a major glycoprotein of Mr 80,000 and three other proteins of Mrs 50,000, 37,000, and 36,000. The nature of p24 protein in secretory granule extracts and the significance of the reduction or absence of Mr 80,000 membrane glycoprotein in this tumor remain to be elucidated.