bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin binding and insulin action in cultured fibroblasts: significant differences between a phosphoglucose isomerase-deficient mutant and the parental strain.
Authors: Wilson C , Peterson S W , Ullrey D B , Kalckar H M
Published: May 2006

The interrelationships of hexose feeding and insulin action were studied in the Chinese hamster fibroblast cell lines 023 and DS-7. The latter, derived from 023 and deficient in phosphoglucose isomerase, has been used to map the metabolic requirements for aldohexose-mediated down-regulation or "curbing" of hexose transport. We have characterized insulin binding and the response to insulin in both cell lines to determine if the insulin-mediated stimulation of transport is similarly dependent on hexose metabolism. DS-7 cells exhibited 5-6 times as many high-affinity insulin binding sites as the parental strain. Apart from this difference, 023 and DS-7 cells showed comparable insulin binding characteristics, which are similar to those observed in other cell types. Insulin at a concentration of 1 microgram/ml (167 nM) was found to stimulate 3-O-methylglucose uptake by approximately equal to 50% in glucose-fed cells of both lines. In neither line did glucose starving significantly alter insulin binding or the insulin-induced stimulation of transport. Feeding with mannose or fructose was found to increase both parameters in 023 cells but had no effect on DS-7 cells. The increase in hexose uptake with the administration of insulin or with glucose starving was shown to be due to an increase in Vmax. Our studies suggest that insulin binding and effect are not regulated by hexose metabolism in the same manner as in the curbing process and insulin induces the recruitment of a transporter pool that is insensitive to hexose curbing.
Expression of the vitellogenin gene in female and male sea urchin.
Authors: Shyu A B , Raff R A , Blumenthal T
Published: May 2006

Expression of vitellogenin, the yolk protein precursor, is strictly regulated during development. In previous studies on a variety of organisms, vitellogenin gene expression has been shown to be restricted to one or two tissues in adult female animals. In this report we show that, in contrast, sea urchin vitellogenin is synthesized in both females and males. To identify sea urchin vitellogenin, we raised antibodies specific for the major yolk protein. We show here that a 155-kDa polypeptide, immunoprecipitable by the antibody to the major yolk protein, is synthesized in the intestines of female and male sea urchins and also in ovaries and testes. This 155-kDa polypeptide is converted to a 195-kDa vitellogenin in each of these tissues; further modification to yield the 180-kDa major yolk protein occurs only in the ovary. We have also identified a vitellogenin cDNA clone and used it to study vitellogenin mRNA production. An abundant 5.1-kilobase mRNA was found in the tissues containing vitellogenin. Our results suggest that vitellogenin may serve the following two functions in sea urchins: its classical role as a yolk protein precursor and an unidentified function required by adults of both sexes.
A phage P1 function that stimulates homologous recombination of the Escherichia coli chromosome.
Authors: Windle B E , Hays J B
Published: May 2006

Recombination between two different defective lacZ genes in the Escherichia coli chromosome (lac- X lac- recombination) was stimulated 2- to 8-fold by prophage P1, depending on the nature of the phage c1 repressor. The P1 BamHI restriction fragment B8 in a lambda-P1:B8 hybrid phage, stimulated lac- X lac- recombination 90-fold in the absence of P1 repressor. A gene necessary for recombination enhancement, designated ref, was localized to one end of B8. Ref expression from lambda-P1:B8 was repressed in trans by a P1 c+ prophage. Two P1 regulatory mutations, bof and lxc, derepressed prophage expression of ref and depressed a prophage function that complemented an E. coli mutant (ssb) deficient in the single-stranded DNA binding protein. Ref stimulation was dependent on preexisting E. coli recombination functions (RecA-RecBC and RecA-RecF). However, other (phage and plasmid) recombination processes involving these functions were not stimulated. ref::Tn5 phages plated and formed lysogens normally. Thus ref appears to be an integral, but not essential, phage gene that stimulates recombination of the host chromosome specifically.
Two Drosophila melanogaster mutations block successive steps of de novo purine synthesis.
Authors: Henikoff S , Nash D , Hards R , Bleskan J , Woolford J F , Naguib F , Patterson D
Published: May 2006

Drosophila melanogaster purine auxotrophs ade2(1) and ade3(1) have been characterized biochemically. The ade2(1) strain is deficient in the fourth step of the de novo purine synthetic pathway catalyzed by phosphoribosylglycinamidine synthase (phosphoribosylformylglycinamide amidotransferase). The ade3(1) strain is deficient in the previous step catalyzed by phosphoribosylglycinamide formyltransferase (GART). The mutation responsible for the slightly leaky ade3(1) phenotype was characterized further. First, the mutant GART polypeptide was found to be of normal size and present at normal levels. Second, the GART-encoding region of the mutant was cloned, inserted into a yeast-Escherichia coli shuttle vector, and used to transform mutant yeast. Transformants showed very slight in vivo activity when compared to wild type, verifying that the mutation is in the GART coding sequence. Lastly, the region of the gene encoding GART activity from mutant and inbred parental strain flies was completely sequenced. A single base transition was found, leading to the substitution of a serine for a highly conserved glycine. These two mutations provide examples of blocks in the de novo purine synthetic pathway in a whole animal.
lac Repressor blocks transcribing RNA polymerase and terminates transcription.
Authors: Deuschle U , Gentz R , Bujard H
Published: May 2006

Operator sequences are essential elements in many negatively controlled operons. By binding repressors, they prevent the formation of active complexes between RNA polymerase and promoters. Here we show that the Escherichia coli lac operator-repressor complex also efficiently interrupts ongoing transcription. This observation suggests a mechanism of action for operators located distal to promoter sequences.
Activation of the endogenous metalloproteinase, gelatinase, by triggered human neutrophils.
Authors: Peppin G J , Weiss S J
Published: May 2006

Triggered human neutrophils degraded denatured type I collagen (gelatin) by releasing and activating the latent metalloenzyme, gelatinase. The ability of the neutrophil to activate this enzyme was significantly, but not completely, inhibited by agents known to inhibit or scavenge chlorinated oxidants generated by the H2O2/myeloperoxidase/chloride system. A direct role for chlorinated oxidants in this process was confirmed by the ability of reagent HOCl to activate the latent enzyme in either the cell-free supernatant or in a highly purified state. Gelatinase activity was also expressed by triggered neutrophils isolated from patients with chronic granulomatous disease. The amount of gelatinolytic activity expressed by the patients' cells was similar to that released by normal neutrophils that were triggered in the presence of antioxidants. Thus, human neutrophils have the ability to activate gelatinase by either an HOCl-dependent process or an uncharacterized oxygen-independent process. The ability of the neutrophil to directly regulate this enzyme suggests an important role for the metalloproteinase in physiologic and pathophysiologic connective tissue metabolism.
Oocyte-specific gene expression: molecular characterization of a cDNA coding for ZP-3, the sperm receptor of the mouse zona pellucida.
Authors: Ringuette M J , Sobieski D A , Chamow S M , Dean J
Published: May 2006

The mouse zona pellucida genes are expressed uniquely during oogenesis and are developmentally regulated in the absence of cell division. Little is known about the mechanisms that control the expression of these germ-line-specific genes that play crucial roles in early mammalian development. We have constructed a lambda gt11 cDNA library from ovarian poly(A)+ mRNA and have isolated clones coding for ZP-3, the mouse sperm receptor. The identity of the clones was confirmed by comparing their DNA sequence with an amino acid sequence obtained from an isolated ZP-3 peptide. The ZP-3 gene is transcribed as a 1.7-kilobase poly(A) mRNA that is detected exclusively in ovarian tissue. This germ-line-specific expression is reflected in the observed hypomethylation of the ZP-3 locus in ovarian but not liver or brain DNAs. The ZP-3 gene is otherwise identically organized in somatic and germ-line DNA where it appears to be present as a low-copy-number or single-copy gene. Despite the fact that the mouse sperm receptor demonstrates species specificity, the ZP-3 cDNA cross-hybridized with DNA from a variety of mammalian species, including rat, rabbit, dog, pig, cow, and human.
Change in antigen specificity of cytotoxic T lymphocytes is associated with the rearrangement and expression of a T-cell receptor beta-chain gene.
Authors: Epplen J T , Bartels F , Becker A , Nerz G , Prester M , Rinaldy A , Simon M M
Published: May 2006

Cloned H-Y-specific murine cytotoxic T lymphocytes, which alter antigen specificity in vitro ("aging"), simultaneously exhibit changes in the T-cell antigen receptor beta-chain rearrangements and respective mRNAs expressed. beta-chain cDNA clones were isolated from a library prepared from mRNA of aged killer T cells. The sequence of the beta-chain variable region element (VAK) was found to be identical with germ-line DNA. Four bases at the beta-chain diversity-joining region (D beta--J beta) junction cannot be explained by known germ-line D beta and J beta elements. These results illustrate that in T-cell clones altered antigen specificity correlates with a switch in productive beta-chain rearrangements of the T-cell receptor. When tested for its expression under physiological conditions, significant levels of VAK mRNA were found in normal lymphocyte populations.
Human serum Cohn fraction IV (alpha-globulin [correction of globin] enriched) inhibits ligand binding at neurotransmitter receptors in human brain.
Authors: Andorn A C , Pappolla M A , Fox H , Klemens F K , Martello P A
Published: May 2006

Human serum proteins are found in significant density in the neuropil in brains of demented individuals. The functional significance of these abnormally distributed proteins has been unknown. We now report that alpha-globulin-enriched fractions of human serum decrease the specific binding of [3H]spiroperidol at its binding sites in postmortem human frontal cortex and caudate. The substances in this serum fraction apparently exert their effect by a direct action on the binding site. Since [3H]spiroperidol labels serotoninergic and dopaminergic among other neurotransmitter receptors, these results suggest that components of human serum inhibit the binding of ligands at neurotransmitter receptors.
Structure of an intermolecular electron-transfer complex: p-cresol methylhydroxylase at 6.0-A resolution.
Authors: Shamala N , Lim L W , Mathews F S , McIntire W , Singer T P , Hopper D J
Published: May 2006

The structure of p-cresol methylhydroxylase [4-cresol:(acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1], a flavocytochrome c, has been determined at 6.0-A resolution. The structure analysis is based on two heavy-atom derivatives with anomalous scattering and 2-fold averaging about a noncrystallographic axis. The molecule is an alpha 2 beta 2 tetramer with a cytochrome subunit of Mr approximately 8500 and a flavoprotein subunit of Mr approximately 49,000. The flavoprotein subunits are tightly packed about the molecular 2-fold axis, whereas the cytochrome subunits are located on the outside of the molecule, each in a depression on the surface of a flavoprotein subunit. The results of this study have led to the following conclusions. The alpha 2 beta 2 quaternary structure of the enzyme is different from alpha beta as originally thought. The orientation of the cytochrome subunit and the surface complementarity of the cytochrome and flavoprotein subunits are clearly defined. The cytochrome subunit is similar in size to other small bacterial cytochromes but probably forms a distinct subclass. The titration (by substrate) behavior of the enzyme and other kinetic properties are rationalized by its quaternary structure.
Identification of processing events in the synthesis of platelet-derived growth factor-like proteins by human osteosarcoma cells.
Authors: Graves D T , Owen A J , Williams S R , Antoniades H N
Published: May 2006

The human osteosarcoma-derived cell line U-2 OS expresses c-sis mRNA and synthesizes platelet-derived growth factor (PDGF)-like proteins. Pulse-chase experiments indicate that proteins of 23 kDa and 180 kDa are synthesized first. The 23-kDa protein undergoes dimerization and proteolysis, giving rise to the 30-kDa dimeric protein secreted by the cells. The 180-kDa protein is proteolytically cleaved in a complex series of steps that give rise to several intracellular species. It is also the likely precursor of high molecular mass PDGF-like or PDGF-associated proteins secreted by these cells. The processing and secretion of the 180-kDa protein is slower than that of the 23-kDa protein. Subcellular fractionation and studies with the antibiotic monensin indicate that the processing events occur in the Golgi-endoplasmic reticulum compartment of U-2 OS cells.
Drosophila histone H2A.2 is associated with the interbands of polytene chromosomes.
Authors: Donahue P R , Palmer D K , Condie J M , Sabatini L M , Blumenfeld M
Published: May 2006

Drosophila chromatin contains two antigenically distinct H2A histones, H2A.1 and H2A.2. Indirect immunofluorescence analyses revealed that anti-H2A.1 binding was distributed throughout polytene chromosomes, whereas anti-H2A.2 binding was interband-specific. Thus, H2A.2 probably contributes to the less compacted structure of interbands. Since each band-interband region is thought to contain a single gene, our results suggest that the distribution of H2A.2 echoes the functional organization of the Drosophila genome. Similar H2A histones occur in eukaryotes ranging from protozoa to mammals. Their placement might be an important determinant of chromatin structure.
Metabolic breakdown of [3H]thymidine and the inability to measure human lymphocyte proliferation by incorporation of radioactivity.
Authors: Bodycote J , Wolff S
Published: May 2006

Quantitative measurement of the incorporation of tritiated thymidine into cultures of phytohemagglutinin-stimulated lymphocytes is routinely used as an indication of the immunocompetence of the cells and of their proliferation. The present experiments show that several components of human blood catabolize nucleosides, including thymidine, extensively. Most of the radioactivity from tritiated thymidine, for example, is quickly rendered unincorporable as the compound is metabolized to thymine and further breakdown products. Thus, cells continue to proliferate without incorporating radioactivity from the medium. Furthermore, variability in the degree of catabolism has been found from person to person, so that neither measurement of the depletion of radioactivity from the medium nor measurement of the amount of label incorporated into the cultures can be used as a quantitative indicator of cell proliferation or immunocompetence.
Rapid evolution of a life history trait
Authors: Hairston Nelson G. , Walton William E.
Published: May 2006

The copepod
Diaptomus sanguineus
begins diapause in permanent ponds in late March as an adaptation to avoid summer fish predation. During a study of copepod populations in two Rhode Island ponds, a severe drought dried one pond killing all fish. The second (control) pond did not dry, and no fish were killed. Before the drought copepods in the two ponds entered diapause on nearly the same date. After the drought, the timing of diapause shifted to later in the year in the pond that had lost its fish, while no shift occurred in the control pond. The direction of this shift in the onset of diapause is that expected had the copepods been released from natural selection for early spring diapause imposed by summer fish predation.
Measurement of low levels of x-ray mutagenesis in relation to human disease.
Authors: Waldren C , Correll L , Sognier M A , Puck T T
Published: May 2006

We previously demonstrated that conventional methods for measurement of mutagenesis in mammalian cells are subject to serious error that causes underestimation of environmental contributions to cancer and genetic disease. This error has been corrected by use of somatic cell hybrids containing a single human chromosome on which the marker genes are carried and by using doses of mutagenic agents so low that little cell killing occurs. This method permits direct measurement of the effects of low doses of radiation and other mutagens without resort to the controversial extrapolation procedure customarily used to estimate effects of doses in the neighborhood of actual human exposures. The new data demonstrate that the true mutagenesis efficiency at the low doses of ionizing radiation that approximate human exposures is more than 200 times greater than those obtained with conventional methods. This methodology also permits evaluation of localized mutations, large and small chromosomal deletions, and nondisjunctional processes and can be used for mutagens that need metabolic activation as well as for cooperatively acting agents. The two opposing classical views that in mammalian cells extrapolation to low doses of x-radiation is linear, on the one hand, or involves a threshold, on the other, are both demonstrated to be incorrect at least for the conditions here considered. The actual curve exhibits a downward concavity so that the mutational efficiency is maximal at low doses. These data may have important implications for human health.
Odorant-binding protein: localization to nasal glands and secretions.
Authors: Pevsner J , Sklar P B , Snyder S H
Published: May 2006

An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.
The fusion-related hydrophobic domain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli.
Authors: Davis N G , Hsu M C
Published: May 2006

Recent work on a prokaryotic membrane protein, gene III protein (pIII) of coliphage f1, showed that polypeptide segments of sufficient hydrophobicity functioned to stop transfer of the polypeptide across the cell membrane: strings of 16 or more hydrophobic amino acids sufficed. A fusion-related hydrophobic domain (FRHD) of Sendai F protein, a sequence of 26 consecutive uncharged residues, has been implicated in the fusion of the viral membrane envelope and the target-cell membrane through a hydrophobic interaction. As it is located on the exterior of the viral membrane, this sequence must be transferred across the host-cell membrane during synthesis. We have inserted either the FRHD or the F protein membrane anchor (the COOH-terminal region of the F protein) into an internal site of a secreted pIII, which lacks its natural membrane anchor. These two hydrophobic sequences behave in the bacteria just as they do in their natural eukaryotic cell host. The F protein membrane anchor functions to stop transfer, conferring a membrane-spanning topology to the F-pIII hybrid protein; however, the FRHD is moved through the cytoplasmic membrane and derivatives carrying this sequence are secreted to the periplasm. We discuss how the FRHD is compatible with passage through the membrane and yet is still able to mediate membrane fusion through a presumed hydrophobic interaction.
Estrogen-induced endogenous DNA adduction: possible mechanism of hormonal cancer.
Authors: Liehr J G , Avitts T A , Randerath E , Randerath K
Published: May 2006

In animals and humans, estrogens are able to induce cancer in susceptible target organs, but the mechanism(s) of estrogen-induced carcinogenesis has not been elucidated. A well-known animal model is the development of renal carcinoma in estrogen-treated Syrian hamsters. Previous work demonstrated the presence of covalent DNA addition products (adducts) in premalignant kidneys of hamsters exposed to the synthetic estrogen, diethylstilbestrol, a known human carcinogen. In the present study, the natural hormone, 17 beta-estradiol, and several synthetic steroid and stilbene estrogens were examined by a 32P-postlabeling assay for their capacity to cause covalent DNA alterations in hamster kidney. Chronic exposure to each of the estrogens tested led to the gradual formation of five chromatographically distinct unusual nucleotides specifically in kidney DNA. Irrespective of the estrogen used, chromatograms exhibited identical mobilities of each of these adducts in seven different systems on PEI-cellulose anion-exchange TLC, in three different conditions on reversed-phase TLC, and in one system on silica gel partition TLC. Therefore, the DNA adducts observed did not contain moieties derived from the structurally diverse estrogens. It is concluded that each of the estrogens induced the binding of the same unknown endogenous compound (or compounds) to target tissue DNA. This novel property of estrogens is postulated to play a key role in hormone-induced malignancy.
Biochemical and electrophysiological evidence of functional vasopressin receptors in the rat superior cervical ganglion.
Authors: Kiraly M , Audigier S , Tribollet E , Barberis C , Dolivo M , Dreifuss J J
Published: May 2006

Binding of radioactive vasopressin--but not of oxytocin--was detected by autoradiography and by labeling of membranes obtained from the rat superior cervical ganglion. In both instances binding could be displaced by V1 (smooth muscle-type) but not by V2 (kidney-type) agonists, indicating that the ganglionic vasopressin receptors are similar to those present on hepatocytes and vascular smooth muscle. In accordance with the V1 character of the receptors, vasopressin activated the turnover of membrane inositol lipids, and this effect was abolished by a structural analogue known to act as a vasopressor antagonist. A possible physiological role of vasopressin was suggested by intracellular recordings obtained from ganglion cells in vitro. Vasopressin induced a reduction in the amplitude of the fast excitatory postsynaptic potential evoked by electrical stimulation of the preganglionic nerve. This reduction in ganglionic transmission was antagonized by the same synthetic structural analogue that blocked the effect of vasopressin on inositol lipids. This study provides evidence for the presence of functional vasopressin receptors in a rat sympathetic ganglion and thus suggests that vasopressin may play a role in peripheral autonomic function.
Integration of staphylococcal phage L54a occurs by site-specific recombination: structural analysis of the attachment sites.
Authors: Lee C Y , Iandolo J J
Published: May 2006

Lysogenization by staphylococcal phage L54a induces the loss of lipase (glycerol ester hydrolase) activity in its host Staphylococcus aureus. The attachment site of the bacterial chromosome (attB) for the phage is at the 3' end of the lipase gene, geh. The DNA fragment containing the attB (base pairs 2620-2637 inclusive) site has been sequenced. We have also cloned and determined the nucleotide sequence of the DNA fragments containing the other three attachment sites--i.e., the attP locus on the circularly permuted phage genome and the attL and attR loci at the left and right ends of the prophage in the lysogenized strain. These results reveal that an 18-base-pair core sequence is common to all four att sites. These data indicate that the crossover point must exist within the core sequence and, further, that integration is site- and orientation-specific. We also localized the viral recombinase gene to a 2.1-kilobase DNA segment extending rightward to the attP site. This region was found to be essential for integration of plasmids containing the attP site.
Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni.
Authors: Davis A H , Blanton R , Rottman F , Maurer R , Mahmoud A
Published: May 2006

Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni. To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals. We report here on the properties of three recombinant clones that derive from developmentally regulated genes. Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli. Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins. The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle. Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs. A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs. Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms. Clone 8-2 contained tandem cDNA inserts. One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms.
Role for topoisomerases in the release of DNA into the detergent-soluble fraction of eukaryotic cells.
Authors: Zhang L H , Mui S C , Todt J C , Strauss P R
Published: May 2006

Detergent-soluble DNA is the fraction (2-4%) of DNA that is released into the supernate upon mild detergent lysis. It is nonmitochondrial in origin. It labels efficiently with deoxy[3H]ribonucleosides and the labeling is prevented by inhibitors of polymerase alpha and ribonucleotide reductase. In previous publications we have characterized detergent-soluble DNA from splenocytes of immunologically activated mice. In this publication we show that incorporation of [3H]thymidine into detergent-soluble DNA is prevented by pretreatment with novobiocin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and teniposide (VM26), three inhibitors of type II topoisomerases. Camptothecin, an inhibitor of type I topoisomerases, also reduces incorporation of [3H]thymidine but only to 50% of control levels. In addition to affecting incorporation of [3H]thymidine, preincubation with the topoisomerase II inhibitors m-AMSA and VM26 alters the amount of DNA recovered in the detergent-soluble fraction. At low concentrations of m-AMSA the amount of detergent-soluble DNA increases somewhat, whereas at higher drug concentrations a marked decrease is observed. Treatment with VM26 results in diminished amounts of DNA being released into the detergent-soluble fraction as well. However, maximal inhibition of detergent-soluble DNA release by VM26 requires the presence of camptothecin. Therefore, we suggest that topoisomerases play an important role in making a small part of lymphocyte chromatin detergent labile. Furthermore, these results are consistent with recent studies demonstrating a role for topoisomerases in yeast replication. Thus, the newly synthesized portion of detergent-soluble DNA may arise as DNA replication intermediates not yet stabilized into mature chromatin.
Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli.
Authors: Ninfa A J , Magasanik B
Published: May 2006

Transcription from nitrogen-regulated promoters, such as glnAp2, requires the glnG gene product, NRI, as well as the rpoN(glnF) gene product, sigma60, and is regulated by the glnL gene product, NRII. We find that in a reaction mixture containing NRI, NRII, and ATP, NRII catalyzes the transfer of the gamma phosphate of ATP to NRI. This covalent modification of NRI occurs concurrently with the acquisition of the ability by the reaction mixture to activate transcription from glnAp2. In the presence of PII, the product of glnB, NRII catalyzes the removal of the phosphate from NRI-phosphate. This reaction occurs concurrently with the loss by the reaction mixture of the ability to activate transcription from glnAp2. On the basis of this evidence, we propose that NRI-phosphate activates transcription from nitrogen-regulated promoters and that the role of NRII is control of the formation and breakdown of NRI-phosphate in response to cellular signals of nitrogen availability.
Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones encoding a galanin precursor.
Authors: Rökaeus A , Brownstein M J
Published: May 2006

The presence of the 29-amino acid peptide galanin and galanin-like immunoreactivity in the peripheral and central nervous system have been established. We describe below the construction of a 1.1-million-recombinant cDNA library derived from poly(A)+ RNA from pig adrenal medulla, rich in galanin-like immunoreactivity, and the isolation and characterization of clones encoding preprogalanin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to galanin-(7-19) and galanin-(22-29) were synthesized and used as hybridization probes to screen the cDNA library. Sequencing of two recombinant plasmids shows that pig galanin mRNA in the adrenal medulla encodes a precursor protein of 123 amino acids that is comprised of a leader sequence, a single galanin sequence, and a 59-amino acid sequence (galanin message-associated peptide) of as yet unknown function. The single galanin sequence, followed by a glycine (the amide donor), is flanked on both sides by pairs of lysine and arginine residues. RNA blot-hybridization analysis of poly(A)+ RNA from pig adrenal medulla reveals a single mRNA of approximately equal to 900-950 bases.
Biologically active precursor for transforming growth factor type alpha, released by retrovirally transformed cells.
Authors: Ignotz R A , Kelly B , Davis R J , Massagué J
Published: May 2006

Retrovirus-transformed rat cells that actively express transforming growth factor type alpha (TGF-alpha) release into the medium a soluble protein of 17-19 kDa that has been identified as a precursor for TGF-alpha. The identification of this protein as a TGF-alpha precursor is based on its recognition by specific antibodies and by the ability of elastase to convert this protein into a 6-kDa polypeptide with the properties of mature TGF-alpha. This TGF-alpha precursor binds to epidermal growth factor/TGF-alpha receptors and activates the receptor-associated tyrosine kinase activity in intact cells. The biological potency of this precursor is not markedly increased by conversion into mature TGF-alpha in vitro. These studies demonstrate the ability of transformed cells to release a TGF-alpha precursor capable of strong mitogenic action in vivo.
Reaction of nucleic acids and cis-diamminedichloroplatinum(II) in the presence of intercalating agents.
Authors: Malinge J M , Leng M
Published: May 2006

The reaction of cis-diamminedichloroplatinum(II) and several synthetic or natural double-stranded polydeoxyribonucleotides has been carried out in the presence of such intercalating agents as ethidium bromide, proflavine, and acridine. After incubation of the reaction mixtures at 37 degrees C for 24 hr, some ethidium or proflavine, but no acridine, molecules are tightly bound to nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or by thin-layer chromatography at basic pH. In the ternary complexes, there is about one tightly bound ethidium (or proflavine) per platinum residue. At 37 degrees C, but not at 4 degrees C, tightly bound ethidium exchanges with free ethidium, whereas platinum residues do not exchange. The binding and the release of tightly bound ethidium are very slow (several hours). It is suggested that in the ternary complexes, nucleic acid-cis-Pt(NH3)2-intercalating agent, a bidentate adduct (guanine-ethidium or -proflavine)-cis-Pt(NH3)2, is formed. No tightly bound ethidium or proflavine is found when cis-diamminedichloroplatinum(II) is replaced by trans-diamminedichloroplatinum(II). Competition experiments between cis-diamminedichloroplatinum(II), poly(dG-dC), and poly(dG)-poly(dC) or poly(dA-dT) show that the presence of ethidium bromide, proflavine, or acridine interferes with the distribution of platinum between the polynucleotides. These results might help to explain the synergism for drugs used in combination with cis-diamminedichloroplatinum(II) and in the design of new chemotherapeutic agents.
Photoaffinity labeling of the gonadotropin receptor with native, asialo, and deglycosylated choriogonadotropin.
Authors: Nishimura R , Raymond M J , Ji I , Rebois R V , Ji T H
Published: May 2006

Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.
Molecular cloning and complete sequence of prion protein cDNA from mouse brain infected with the scrapie agent.
Authors: Locht C , Chesebro B , Race R , Keith J M
Published: May 2006

The prion protein (PrP) is a scrapie-associated fibril protein that accumulates in the brains of hamsters and mice infected with the scrapie agent, and also in the brains of persons affected with kuru or Creutzfeldt-Jakob disease. It has been previously proposed that PrP could be either the primary transmissible agent of scrapie or a secondary component involved in the pathogenesis of scrapie. At present, the second possibility seems more likely, for the PrP-specific mRNA is present in both infected and uninfected brains. We have isolated and sequenced the complete PrP-specific cDNA from mRNA isolated from infected mouse brains. Comparison of the mouse PrP with the hamster PrP reveals a high homology in the amino acid sequence and the presence of a conserved octapeptide repeated four times, whose function is unknown at present. Structural features are discussed and compared with other proteins. Except for its homology with the hamster PrP, mouse PrP has no significant homology to any known protein sequence, including neurofilaments, neuropeptides, and amyloid proteins of Alzheimer disease. Some features of the PrP, however, are similar to structures found in aggregating proteins, such as the wheat glutenin, keratin, and collagen.
Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA.
Authors: Evans C T , Ledesma D B , Schulz T Z , Simpson E R , Mendelson C R
Published: May 2006

A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene.
DNase I-hypersensitive sites in the 5'-flanking region of the rat serum albumin gene: correlation between chromatin structure and transcriptional activity.
Authors: Babiss L E , Bennett A , Friedman J M , Darnell J E
Published: May 2006

As tested by DNase I digestion, the chromatin structure in several regions 5' to the rat serum albumin gene varies in tissues and cell lines that differ in transcription rate of this gene. Three DNase I-hypersensitive regions were found in hepatocyte nuclei but not in kidney cell nuclei. The sites were approximately 2.8 kbp (site 1), 0.2 kbp (site 2), and 0.05 kbp (site 3) upstream from the cap site of the gene. In rat fetal liver tissue and rat hepatoma cell lines (FaO, C2, and C2-rev7), as well as in cultured primary hepatocytes where the rate of albumin gene transcription is lower than in adult liver, hypersensitive site (HSS) 1 was absent while sites 2 and 3 were present. In addition, the C2 cell line, which does not express albumin mRNA, contains a different HSS at position -1.5 kbp. Factors (proteins) bound to sites 2 and 3 may allow cell-specific transcription, but the additional factor interaction at site 1 could be required for a maximal rate of albumin gene transcription.
Reaction of DNA with chemically or enzymatically activated mitomycin C: isolation and structure of the major covalent adduct.
Authors: Tomasz M , Chowdary D , Lipman R , Shimotakahara S , Veiro D , Walker V , Verdine G L
Published: May 2006

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2'' beta,7''-diaminomitosen-1'' alpha-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1'' beta stereoisomer of 2 (Structure 3), and 10''-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC).poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and O6 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.
U1, U2, and U6 small nuclear ribonucleoproteins (snRNPs) are associated with large nuclear RNP particles containing transcripts of an amplified gene in vivo.
Authors: Sperling R , Spann P , Offen D , Sperling J
Published: May 2006

Nuclear ribonucleoprotein (RNP) complexes that contain intact transcripts of the amplified gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells, have been released from nuclei of Syrian hamster cells as large particulate structures that sediment at the 200S region in a sucrose gradient. By the technique of RNA hybridization, we have shown that U1, U2, and U6 small nuclear RNAs (snRNAs) cosediment with the large RNP particles in the sucrose gradients. Autoimmune sera from systemic lupus erythematosus and mixed connective tissue disease patients, characterized as anti-(U1)RNP, have further been shown to immunoprecipitate CAD RNA along with U1 and U2 snRNAs from the fractionated nuclear 200S RNP particles. We conclude that U1, U2, and U6 snRNPs are integral constituents of the 200S RNP particles. The requirement of snRNPs for RNA processing that evidently occurs on RNP particles has been recently demonstrated. Our results thus suggest that the 200S RNPs are structurally and functionally close to the native particles on which RNA processing occurs.
The modulation of Hb F synthesis in adult erythroid progenitor (burst-forming unit) cultures reflects changes in gamma-globin gene transcription and chromatin structure.
Authors: Groudine M , Peretz M , Nakamoto B , Papayannopoulou T , Stamatoyannopoulos G
Published: May 2006

Fetal hemoglobin production can be reactivated in vivo in adult persons with various hemoglobinopathies and other hemopoietic disorders, and in cultures of adult erythroid progenitors. We show that the activation of Hb F in adult cells is transcriptional in nature and is accompanied by the appearance of DNase I-hypersensitive sites and undermethylation of Hpa II sites 5' to the gamma-globin genes. Production of Hb F in culture is strongly modulated by the environment, and the repression of Hb F synthesis by specific culture conditions has been reported. By nuclear runoff, chromatin, and methylation analyses, we show that this inhibition of Hb F production in vitro is at the level of transcription with the concomitant loss of characteristic gamma hypersensitive sites and methylation of gamma Hpa II sites. These data indicate, first, that the organization of globin chromatin of adult cells that produce fetal hemoglobin resembles that of fetal erythroid cells and, second, that this organization switches from a fetal to an adult pattern in response to changes in the environment of the erythroid cells.
Synthesis of nerve growth factor mRNA and precursor protein in the thyroid and parathyroid glands of the rat.
Authors: Dicou E , Lee J , Brachet P
Published: May 2006

Nerve growth factor (NGF) mRNAs were detected in the thyroid and parathyroid glands by hybridization to a preproNGF and a NGF-beta cDNA probe. The thyroid NGF transcript was identical in size to that found in the mouse submaxillary gland. Mature NGF-beta was not detected in the thyroid tissue either by immunoprecipitation or by immunohistochemical methods. In contrast, an antiserum directed against the predicted precursor protein sequence immunoprecipitated a protein of Mr 30,000-31,000 in thyroid extracts. A protein of identical molecular weight was also detected by this serum in the mouse submaxillary gland extracts. In vitro translation of hybrid-selected NGF RNAs detected two peptides of Mrs 35,000 and 28,000. These peptides closely correspond to the proposed translation products of the preproNGF mRNA sequence, suggesting that two different initiation codons can be utilized, at least in vitro. The proNGF antiserum also cross-reacted with the in vitro synthesized Mr 35,000 peptide. The antigenic determinant recognized by the precursor-specific antibody was localized to the convoluted granular tubules of the mouse submaxillary gland, known to be the site of synthesis of the NGF-beta. We present evidence for the localization of unprocessed proNGF in the parathyroid gland and in some parafollicular cells of the thyroid.
DNA sequence adjacent to and specific for the 1.672 g/cm3 satellite DNA in the Drosophila genome.
Authors: Donnelly R J , Kiefer B I
Published: May 2006

The Drosophila genome contains nearly 2.8 X 10(4) kilobases of satellite DNA. This simple sequence satellite DNA is contained within transcriptionally inactive heterochromatin that is distributed among all chromosomes with a concentration at the centromeres and along the length of the Y chromosome. To investigate the relationship of the satellite DNA with the surrounding sequences, we have isolated a satellite junction sequence that is repetitive and specifically adjacent to the 1.672 g/cm3 satellite DNA. It is conserved between strains of Drosophila melanogaster and localized to the chromocenter of polytene chromosomes. The characteristics of this sequence suggest a functional role involving the specific organization of large regions of chromosomes.
Molecular characterization and phylogenetic studies of a wound-inducible proteinase inhibitor I gene in Lycopersicon species.
Authors: Lee J S , Brown W E , Graham J S , Pearce G , Fox E A , Dreher T W , Ahern K G , Pearson G D , Ryan C A
Published: May 2006

A gene coding for proteinase inhibitor I, whose expression is induced in tomato leaves (Lycopersicon esculentum L. var. Bonny Best) in response to wounding or insect attacks, was isolated from a genomic library and characterized. The nucleotide sequence revealed that the gene is complete and encodes the sequence of an inhibitor I cDNA that was previously isolated from a cDNA library prepared from wound-induced mRNA from tomato leaves. This gene is located 13.1 kilobase pairs (kbp) upstream from an inhibitor II gene. The wound-inducible gene is interrupted by two intervening sequences of 445 and 404 bp, situated within the codons of amino acids 17 and 47, respectively, of the open reading frame. In addition to the presence of putative regulatory sequences, TATAAA and CCACT, two copies of an imperfect direct repeat approximately 100 bp long were identified in the 5'-flanking region. Phylogenetic comparisons of wound-inducible inhibitor I genes within the genomes of various Lycopersicon species revealed that the repeat is found in seven ancestral species of tomato.