bluuebunny/crossref_metadata_2025 huggingface dataset:
Perturbation of maintenance and de novo DNA methylation in vitro by UVB (280-340 nm)-induced pyrimidine photodimers.
Authors: Becker F F , Holton P , Ruchirawat M , Lapeyre J N
Published: May 2006

The effect of pyrimidine photodimers on transmethylation reactions catalyzed by a highly purified rat liver DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) that exhibits maintenance and de novo methylation activities was studied in vitro, using the viral substrates M13 mp9 replicative form (RF) DNA and the hemimethylated analog formed from primed synthesis of phage DNA in the presence of 2'-deoxy-5-methylcytidine 5'-triphosphate. These DNAs were irradiated with UVB (280-340 nm) at 900-3600 J/m2 in the presence of the triplet-state sensitizers acetone or 3-dimethylaminopropiophenone. Under these conditions of irradiation, which approximate solar UV, pyrimidine cyclobutane photodimers were introduced without producing any evidence of single-strand breaks or alkali-sensitive sites [i.e., no (6-4)pyrimidine-pyrimidone photoproducts]. This was confirmed by gel analysis, a T4 UV endonuclease nicking assay specific for cyclobutane-type dimers, and HPLC analysis of the photoproducts. The methylation of irradiated templates by DNA methyltransferase was inhibited in an approximately linear fashion as a function of increasing UVB dose. This inhibition was correlated with the number of lethal photoproducts detected by the simultaneous measurement of the surviving fraction of infectious phage DNA. For approximately the same number of pyrimidine cyclobutane photoproducts introduced, de novo methylation activity was approximately 2-fold more sensitive than the maintenance mode of methylation. The ability of these putatively carcinogenic, pyrimidine photoproducts to inhibit DNA methylation suggests a common mechanism of action with several chemical carcinogens that are known to modify bases.
Structures of defective P transposable elements prevalent in natural Q and Q-derived M strains of
Drosophila melanogaster
Authors: Sakoyama Yasuhiko , Todo Takeshi , Ishiwa-Chigusa Sadao , Honjo Tasuku , Kondo Sohei
Published: May 2006

Several DNA sequences with homology to the complete 2.9-kilobase (kb) P element from a P strain in the United States were isolated and characterized from two
Drosophila melanogaster
strains collected on Chichi Jima, an island 1000 km south of Tokyo. Except for a missing central region and trivial unsequenced regions of 38 base pairs, the 2.1-kb element isolated from a Q strain had the same DNA sequence as that of the complete P element. Seven other elements cloned from genomic DNAs of the Q strain and a Q-derived M strain all possessed the same restriction sites as those of the 2.9-kb P element except for one deleted region in each element. The finding of sequence conservation in P elements have had a common ancestor relatively recently. Thus, it is suggested that the P element family was a recent invader of the species. By contrast, no complete P element was found in these Japanese strains so far as surveyed, indicating the possibility that P elements in the Chichi Jima population are almost all defective. The implication of this possibility is discussed in relation to the uniqueness of the population on Chichi Jima where Q strains predominate and no P strains have yet been found.
Carboxyl-terminal domain of the Epstein-Barr virus nuclear antigen is highly immunogenic in man.
Authors: Milman G , Scott A L , Cho M S , Hartman S C , Ades D K , Hayward G S , Ki P F , August J T , Hayward S D
Published: May 2006

The carboxyl-terminal one-third of the Epstein-Barr virus nuclear antigen (EBNA-1) encoded by the BamHI restriction fragment K was synthesized in Escherichia coli by use of a high-expression plasmid. The resultant 28-kDa EBNA fusion polypeptide, comprising 5-10% of the total soluble bacterial protein, was purified to apparent homogeneity by phosphocellulose and hydroxylapatite column chromatography. Both rabbit monospecific antibodies and mouse monoclonal antibodies against 28-kDa EBNA gave nuclear immunofluorescence staining on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines and recognized the appropriate intact EBNA polypeptide bands on immunoblots. An ELISA with the purified 28-kDa EBNA as antigen was used to quantitate anti-EBNA antibody in human serum samples. The ELISA method was approximately 100-fold more sensitive than the classical anticomplement immunofluorescence assay. Anti-EBNA antibody was detected in sera from 100% of normal individuals who were seropositive for the viral capsid antigen, and low anti-EBNA titers were detected in serum from most patients with acute infectious mononucleosis. The assay gave the expected pattern of titers in sera from patients with rheumatoid arthritis, Burkitt lymphoma, or nasopharyngeal carcinoma, thus confirming the validity of this purified reagent for assessing EBNA antibody status. Approximately 10% of normal individuals and rheumatoid arthritis patients had anti-EBNA titers as high as those seen in nasopharyngeal carcinoma patients. In these high-titer individuals, greater than 1% of the total IgG are antibodies that recognize 28-kDa EBNA, which indicates that the carboxyl-terminal domain of EBNA is highly immunogenic.
Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+,K+-ATPase activity.
Authors: Angel I , Hauger R L , Luu M D , Giblin B , Skolnick P , Paul S M
Published: May 2006

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37 degrees C) resulted in a time-dependent decrease in specific (+)-[3H]amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-[3H]amphetamine binding, whereas incubation with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-[3H]amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-[3H]amphetamine binding, suggesting the involvement of Na+,K+-ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na+,K+-ATPase activity and the number of specific "high-affinity" binding sites for [3H]ouabain, and a good correlation was observed (r = 0.89; P less than 0.02) between the glucose-stimulated increase in (+)-[3H]amphetamine and [3H]ouabain binding. Similar increases in (+)-[3H]amphetamine binding, [3H]ouabain binding, and Na+,K+-ATPase activity were observed in the hypothalamus after parenteral administration of glucose to rats. The administration of anorectic doses of amphetamine (0.1-5.0 mg/kg of body weight) also increased Na+,K+-ATPase activity in the hypothalamus. These data suggest that the (+)-[3H]amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na+,K+-ATPase activity, and the latter may be involved in the "glucostatic" regulation of appetite.
Changes in plasma proenkephalin peptide F and catecholamine levels during graded exercise in men.
Authors: Kraemer W J , Noble B , Culver B , Lewis R V
Published: May 2006

Proenkephalin peptide F immunoreactivity, epinephrine, and norepinephrine were measured in the plasma of endurance-trained and untrained male subjects riding on a bicycle ergometer at 28%, 54%, 83%, and 100% of maximum oxygen consumption (VO2). At rest the trained group had peptide F levels almost twice the level of the untrained group, whereas all other variables measured were the same. The maximum epinephrine and norepinephrine levels were found at 100% exercise intensity, with a precipitous drop in the levels at 5 min of recovery. In contrast, the peptide F immunoreactivity reached a maximum at 5 min of recovery and was still substantially above the initial level after 15 min of rest. In addition, the trained subjects showed another peak of peptide F immunoreactivity at 54% VO2max. Possible explanations for the different patterns of catecholamine and peptide F levels are presented.
Inositol 1,4,5-trisphosphate: a possible chemical link in excitation-contraction coupling in muscle.
Authors: Vergara J , Tsien R Y , Delay M
Published: May 2006

The role of inositol 1,4,5-trisphosphate (InsP3) in excitation-contraction coupling in skeletal muscle was investigated by several methods. The following results were obtained. InsP3 is released by electrical stimulation of muscles. Exogenous InsP3 releases calcium from skinned muscle fibers at relatively high doses under normal conditions but does so at very low concentrations when blockers of the InsP3 5-phosphatase are present. Blockers of InsP3 release are effective blockers of calcium transients elicited by electrical stimulation of muscle fibers. It is proposed that InsP3 acts as a chemical second messenger between transverse (T)-tubular membrane depolarization and calcium release from the sarcoplasmic reticulum in skeletal muscle.
Preferential rearrangement of the immunoglobulin kappa chain joining region J kappa 1 and J kappa 2 segments in mouse spleen DNA.
Authors: Nishi M , Kataoka T , Honjo T
Published: May 2006

The V kappa-KpnI family, which constitutes approximately equal to 36% of mouse kappa chain variable region gene (V kappa) segments, conserves the Kpn I site (G-G-T-A-C-C) at the position corresponding to residues 35-37. Using this cleavage site, we were able to assess the relative recombination frequency of the kappa chain joining region gene (J kappa) segments in mouse spleen DNA. The J kappa 1 and J kappa 2 segments were used 2- to 5-fold more frequently than were the J kappa 4 and J kappa 5 segments. The J kappa 3 segment was shown to be incapable of recombining with the V kappa segment. The relative recombination frequency of the J kappa segments did not change significantly by lipopolysaccharide stimulation of normal mouse spleens. The relative frequency of the J kappa usage was unaltered in immune disorders such as in nude (nu/nu), MRL (lpr/lpr), and BXSB mice. Seven V kappa-KpnI-J kappa 1 clones were isolated, and their nucleotide sequences were determined. Two of them were derived from the identical germ-line V kappa segment but differed in the nucleotide sequence of the V-J junction. The maximal number of V kappa germ-line segments was estimated to be less than 300 by statistical calculation.
DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region.
Authors: Hediger M A , Johnson D F , Nierlich D P , Zabin I
Published: May 2006

The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is the translation initiation codon. A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints. A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing. An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA.
Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris(diphenylphenanthroline)cobalt(III).
Authors: Barton J K , Raphael A L
Published: May 2006

The chiral complex tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), lambda-Co(DiP)3(3+), binds to and, with photoactivation, cleaves left-handed DNA helices, thereby providing a unique molecular probe for local DNA conformation. We have mapped the specific left-handed sites where lambda-Co(DiP)3(3+) cleaves in the plasmids pBR322 and pLP32, which is the derivative of pBR322 containing a Z-form d(C-G)16 insert. For pLP32, a primary cleavage is at the insert; for native pBR322, cleavage occurs at four discrete sites: 1.45, 2.3, 3.3, and 4.2 kilobase pairs. These sites correspond to segments of alternating purine-pyrimidines. Moreover, these positions map to the ends of the three distinct coding regions in pBR322: the tetracycline-resistance gene, the origin of replication, and either end of the ampicillin-resistance (beta-lactamase) gene. The locations of these left-handed segments suggest to us that Z-DNA might serve as a conformational punctuation mark to demarcate the ends of genes.
A molecular dynamics simulation of double-helical B-DNA including counterions and water.
Authors: Seibel G L , Singh U C , Kollman P A
Published: May 2006

We present the results of an atomic level molecular dynamical simulation of a 5-base-pair fragment of double-helical DNA with inclusion of water and sodium counterions and a complete description of their electrostatic interactions. The shape of the double helix is preserved throughout the simulation, and the helix repeat is calculated to be 10.0, in reasonable agreement with experimental results. The most flexible conformational angles in the structure are the glycosidic angle and the sugar pucker.
One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells.
Authors: Protić-Sabljić M , Kraemer K H
Published: May 2006

We have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. We studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines (D0 = 56 J X m-2) than in the other human cell lines tested (normal, ataxia-telangiectasia, Lesch-Nyhan, retinoblastoma)(D0 = 680 J X m-2)(D0 is the dose that reduces the percentage of CAT activity by 63% along the exponential portion of the dose-response curve). The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.
Leukotriene C4 production by murine mast cells: evidence of a role for extracellular leukotriene A4.
Authors: Dahinden C A , Clancy R M , Gross M , Chiller J M , Hugli T E
Published: May 2006

The glutathione-containing leukotriene C4 (LTC4) is a major mediator of smooth muscle contraction and is released by mast cells when antigen interacts with cell-bound IgE. Antigen-stimulated mast cells undergo phospholipase activation. We report a pathway of LTC4 production by mast cells that does not require phospholipase activation but depends on the interaction of activated neutrophils with unstimulated mast cells, using as an intermediate extracellular leukotriene A4 (LTA4). The epoxide LTA4 is released by neutrophils and, together with leukotriene B4 and 5-hydroxyeicosatetraenoic acid, constitutes the major lipoxygenase metabolites found in supernatants of stimulated neutrophils. Five minutes after activation of neutrophils by calcium ionophore A23187 we measured 136 pmol of extracellular LTA4 per 10(7) neutrophils (range 40-300, n = 7) by trapping the epoxide with alcohols. Therefore, we conclude that LTA4 is not just an intracellular leukotriene precursor but is released as a lipoxygenase metabolite. LTA4 is known to be stabilized by albumin and is efficiently converted by mast cells into LTC4 even at low LTA4 concentrations. The LTA4 complexed to albumin is converted into LTC4 rapidly and completely within 10-15 min. More than 50% of the LTA4 presented to mast cells is metabolized to LTC4 at concentrations of LTA4 between 0.2 and 2 nmol of LTA4 per 10(7) mast cells. This observation establishes a potential physiologic role for extracellular LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes.
Direct cross-linking of 125I-labeled glucagon to its membrane receptor by UV irradiation.
Authors: Iwanij V , Hur K C
Published: May 2006

125I-labeled glucagon was directly crosslinked to its receptor in isolated liver plasma membranes and on the surface of intact hepatocytes, by using a UV irradiation procedure. This investigation resulted in the identification of a glucagon-receptor complex of apparent Mr 62,000. The specificity of labeling was shown by the interference of unlabeled hormone at physiological concentration with incorporation of radioactive glucagon into the 62,000 Mr species. The receptor behaved as a typical integral membrane protein: it was not released by extraction with lithium diiodosalicylate or at basic pH but was solubilized by digitonin treatment. Reduction of the receptor polypeptide with dithiothreitol resulted in a decrease in its electrophoretic mobility, suggesting the presence of intramolecular disulfide bonds. Soluble glucagon-receptor complexes adsorbed to Con A-Sepharose and could be eluted with methyl alpha-D-mannoside, indicating that the receptor molecule is a glycoprotein. Treatment of glucagon-labeled liver plasma membrane with endoglycosidase F resulted in the appearance of four intermediate species, indicating that glucagon receptor contains at least four N-linked oligosaccharide chains.
Isolation and characterization of factors from wheat germ that exhibit eukaryotic initiation factor 4B activity and overcome 7-methylguanosine 5'-triphosphate inhibition of polypeptide synthesis.
Authors: Lax S , Fritz W , Browning K , Ravel J
Published: May 2006

Three highly purified preparations (preparations I, II-1, and II-2) have been obtained from wheat germ and shown to support in vitro polypeptide synthesis directed by capped or uncapped mRNAs in a eukaryotic initiation factor 4B (eIF-4B)-deficient system. The three preparations differ, however, in polypeptide composition and in the ability to overcome the inhibitory effect of 7-methylguanosine 5'-triphosphate (m7GTP) on in vitro polypeptide synthesis. Preparation I contains two polypeptides (Mr = 80,000 and 28,000), which are present in a 1:1 molar ratio and are associated in a high molecular weight complex. Preparation II-1 contains two major polypeptides (Mr = 220,000 and 26,000) and preparation II-2 also contains two major polypeptides (Mr = 110,000 and 26,000). Preparations II-1 and II-2 are high molecular weight complexes; neither contains detectable amounts of a Mr 80,000 or a Mr 50,000 component. Preparations II-1 and II-2 both overcome m7GTP inhibition, whereas preparation I does not. These findings raise several questions with regard to the identity of eIF-4B and its relationship to cap recognition factors.
Hypermutation at the immunoglobulin heavy chain locus in a pre-B-cell line.
Authors: Wabl M , Burrows P D , von Gabain A , Steinberg C
Published: May 2006

Most cells in the well-known pre-B-lymphocyte line 18-81 have correctly assembled genes for both alleles at the immunoglobulin heavy chain locus. Only one allele is "active"; the other, "silent" allele contains an amber termination codon. The rate of reversion of this amber codon was determined to be 0.3-1 X 10(-4) per cell generation. This high rate is termed hypermutation.
Identification of a single chromosome in the normal human genome essential for suppression of hamster cell transformation.
Authors: Stoler A , Bouck N
Published: May 2006

Normal human fibroblasts were fused to carcinogen-transformed baby hamster kidney (BHK) cells and found to be able to suppress the anchorage-independent transformed phenotype of the hamster cells. This suppression was not due to interspecies incompatibility, for transformation could be effectively expressed in hybrids if either the human or the BHK parent had initially been transformed by a dominantly acting viral genome. Upon growth of suppressed hybrids, loss of human chromosomes was accompanied by the re-expression of transformation. Karyotype analysis indicated that only human chromosome 1 was retained in all hybrids that were suppressed and was lost in all hybrids in which transformation was re-expressed. Cytological evidence for the presence or absence of chromosome 1 was confirmed by electrophoretic identification of the human isozyme for phosphoglucomutase 1. Clones re-expressing transformation were isolated from two suppressed hybrids and in both cases loss of suppression was accompanied by the loss of human chromosome 1. Thus, the maintenance of suppression in these cross-species hybrids appears to require the continued presence of normal human chromosome 1. These findings raise the possibility that the frequent involvement of human chromosome 1 in potentially inactivating aberrations in human tumors may reflect a suppressor role for this chromosome in human malignancy.
Characterization of octopamine-sensitive adenylate cyclase: elucidation of a class of potent and selective octopamine-2 receptor agonists with toxic effects in insects.
Authors: Nathanson J A
Published: May 2006

Octopamine-2 receptors, associated with activation of adenylate cyclase, mediate a number of the important hormonal and neurotransmitter functions of octopamine in invertebrates. By utilizing the highly enriched octopamine-sensitive adenylate cyclase present in the firefly light organ, it has been possible to pharmacologically characterize octopamine-2 receptors and to define a new class of highly potent and selective octopamine-2 agonists. At low concentrations, these substituted phenyliminoimidazolidines stimulate light emission when injected into fireflies. At somewhat higher concentrations, these compounds, when ingested by tobacco hornworms, cause disruption of motor and feeding behavior, leading to insect death. The effects of these compounds are markedly potentiated by phosphodiesterase inhibitors and mimicked by other activators of octopamine-sensitive adenylate cyclase, including octopamine itself. Because octopamine-2 receptors appear to be present primarily in invertebrates, these findings, together with other data, raise the possibility that potent and selective octopamine agonists could be useful as insect toxins with low toxicity in vertebrates.
Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.
Authors: Padan E , Sarkar H K , Viitanen P V , Poonian M S , Kaback H R
Published: May 2006

The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a normal complement of permease molecules, as determined by immunoadsorption assays. Interestingly, although substitution of histidine-205 or histidine-322 by arginine results in the loss of ability to catalyze active lactose transport, permease molecules with arginine at residue 322 appear to facilitate downhill lactose movements at high concentrations of the disaccharide. The results provide strong support for the contention that histidine residues in the lac permease play an important role in the coupling between lactose and proton translocation.
Electron transfer-driven ATP synthesis in
Methanococcus voltae
is not dependent on a proton electrochemical gradient
Authors: Crider B. P. , Carper S. W. , Lancaster J. R.
Published: May 2006

Intracellular ATP levels in whole cells of
Methanococcus voltae
respond to electron transfer coupled to methanogenesis. ATP synthesis can also be induced by an artificially imposed transmembrane electrical potential [formed by electrogenic movement outwards of potassium (induced by valinomycin) or of protons (induced by an uncoupler], or by a pH gradient (acid outside). These results implicate the existence of a reversible ATPase coupled to electrogenic movement of an ion(s) other than potassium or proton, and that ionophores are competent to catalyze ion movement across the cytoplasmic membrane of this organism (which is the sole membrane structure in this species). ATP synthesis driven by electron transfer is, however, insensitive to the addition of such ionophores. These results indicate that although cells possess an ion-translocating ATPase (possibly involved in the maintenance of internal ionic composition homeostasis), methanogenesis-driven ATP synthesis does not involve the intermediacy of a transmembrane ion gradient. Primarily because methane formation has been previously demonstrated to involve true electron transfer, substrate-level phosphorylation (at least in analogy to other systems) has been generally ruled out. The results presented here suggest that at least one methanogenic bacterium may use a direct linkage of ATP synthesis to electron transfer.
Heat shock increases the synthesis of the poly(A)-binding protein in HeLa cells.
Authors: Schönfelder M , Horsch A , Schmid H P
Published: May 2006

When HeLa cells are shifted from 37 degrees C to 45 degrees C, the synthesis of two proteins increases. Their approximate molecular masses are 73 kDa [heat shock protein 73 (hsp73)] and 87 kDa (hsp87), respectively. One of them, the hsp73, shows a specific affinity for poly(A). This protein is identical with a protein regularly found associated with translatable mRNAs in all vertebrate cells. It is well characterized by its high affinity to the poly(A) sequence of polyribosomal mRNA, and it occurs free in cytoplasm. hsp73 and the poly(A)-binding protein have the same isoelectric point and molecular size. The peptide analysis indicates that they are identical.
Autocrine growth stimulation of a human T-cell lymphoma line by interleukin 2.
Authors: Duprez V , Lenoir G , Dautry-Varsat A
Published: May 2006

The ability of tumor cells to produce and to respond to their own growth factor (autocrine secretion) may be of importance for their growth. We describe a human tumor cell line regulated by an autocrine secretion of the growth factor interleukin 2 (IL-2). This T-lymphocyte cell line, IARC 301, was established from a patient with a T-cell lymphoma in the absence of any added specific growth factor. It constitutively expresses biologically functional high-affinity cell-surface receptors for IL-2 as shown by the binding of both radiolabeled purified IL-2 and monoclonal antibodies to IL-2 receptors. In addition, it synthesizes IL-2, which is bound to cell surface receptors. Monoclonal antibodies directed against either IL-2 or the IL-2 receptor block IARC 301 cell growth. These findings demonstrate that the proliferation of this tumor cell line is mediated by an autocrine pathway involving endogenous IL-2 production and its binding to cell surface receptors.
Chloroplast DNA diversity is low in a wild plant, Lupinus texensis.
Authors: Banks J A , Birky C W
Published: May 2006

Chloroplast DNA diversity was measured in an annual flowering plant, Lupinus texensis. Individual plants were collected from 21 local populations throughout the range of the species in Texas. Chloroplast DNA was isolated separately from each plant and digested with seven restriction enzymes. The most common form of the 150-kilobase-pair genome was cut at 134 sites, so that about 0.5% of the base pairs in the genome were sampled. Of the 100 plants examined, 88 had identical restriction fragment patterns. Three variant forms were found in different local populations. Two, represented in single plants, differed from wild type in the presence or absence of single restriction sites. The third variant was fixed in one of the local populations; it had lost a restriction site and also had a deletion of approximately equal to 100 base pairs. The data suggest that chloroplast DNA in this plant is much less polymorphic than mitochondrial DNA from animals and is probably less polymorphic than nuclear genes in the same plant or in animals.
Alloreactive cytolytic T-cell clones preferentially recognize conformational determinants on histocompatibility antigens: analysis with genetically engineered hybrid antigens.
Authors: Arnold B , Horstmann U , Kuon W , Burgert H G , Hämmerling G J , Kvist S
Published: May 2006

Hybrid genes were constructed for the localization of allodeterminants on murine class I antigens recognized by antibodies and cytolytic T lymphocytes. By using deletion subclones of the H-2Kd and H-2Kk genes, homologous regions were exchanged between the two alleles. The altered genes were introduced and expressed in mouse fibroblast and fibrosarcoma cells. Cells expressing hybrid antigens were analyzed with 29 monoclonal anti-H-2Kd and anti-H-2Kk antibodies and with 150 short-term alloreactive cytolytic T-cell clones. When only the first or only the second amino-terminal domain was exchanged, most T cells and 60% of the antibodies lost their reactivity to the H-2K antigen. No T-cell clone was directed against the third extracellular domain, whereas three antibodies could bind to this domain. This implies that nearly all determinants essential for a cytolytic T-cell response or for antibody binding lie on the two external domains and are conformational structures generated by the interaction of these two domains.
Synthesis and secretion of human epidermal growth factor by Escherichia coli.
Authors: Oka T , Sakamoto S , Miyoshi K , Fuwa T , Yoda K , Yamasaki M , Tamura G , Miyake T
Published: May 2006

A synthetic gene for human epidermal growth factor (hEGF) was joined to a sequence encoding the signal peptide of Escherichia coli alkaline phosphatase. This hybrid gene was placed under the control of the alkaline phosphatase gene (phoA) promoter in a recombinant plasmid, which was used to transfect E. coli. The hybrid protein that was expressed in host cells under conditions of phosphate limitation was processed accurately during the secretion process, and mature hEGF was recovered in the periplasmic fraction. On the other hand, no EGF was detected in the periplasmic space when the synthetic hEGF gene was not accompanied by the phoA signal sequence.
Changes in
22
Na influx and outflux in
Daphnia magna
(Straus) as a function of elevated Al concentrations in soft water at low pH
Authors: Havas Magda , Likens Gene E.
Published: May 2006

The effects of aluminum (Al) on sodium (Na) regulation by the freshwater crustacean
Daphnia magna
were determined.
22
Na influx and outflux experiments were conducted in soft water adjusted to pH 4.5, 5.0, and 6.5 (reference pH) with either ambient (0.02 mg/liter) or high (1.02 mg/liter) concentrations of total Al. The results indicate that Al toxicity was pH dependent. Aluminum increased the rate of morbidity of
D. magna
at pH 6.5, had no additional effect to those of hydrogen ions (H
+
) at pH 5.0, and reduced the rate of morbidity at pH 4.5. Both H
+
and total Al concentrations interfered with Na regulation, although it was possible to distinguish between their respective effects by using
22
Na. At pH 6.5, Al decreased
22
Na influx (by 46%) and increased
22
Na outflux (by 25%), which led to a net loss of Na. At pH 5.0, Al reduced
22
Na influx (by 58%) but had no additional effect to that of H
+
on
22
Na outflux. At pH 4.5,
22
Na influx was significantly inhibited (by 73%) compared with the reference pH 6.5 treatment even in the absence of Al. Aluminum decreased
22
Na outflux (by 31%) at pH 4.5, which reduced the net loss of Na and temporarily prolonged survival of the daphnids. These results indicate that Al affects both
22
Na influx and outflux in
D. magna
. The lower rate of Na uptake may involve a denaturation of the enzyme responsible for the active uptake of Na. At pH 6.5, the increased outflux of
22
Na may be due to either increased membrane permeability or increased renal losses (or both). At pH 4.5, the reduced outflux of
22
Na resembles the amelioration that occurs in the presence of elevated Ca concentrations.
IgM RNA switch from membrane to secretory form is prevented by adding antireceptor antibody to bacterial lipopolysaccharide-stimulated murine primary B-cell cultures.
Authors: Chen-Bettecken U , Wecker E , Schimpl A
Published: May 2006

Bacterial lipopolysaccharide (LPS) induces proliferation of resting primary murine B lymphocytes and their differentiation into Ig-secreting cells. This is accompanied by an increase in the rate of Ig gene transcription and the accumulation of mu heavy chain secretory mRNA. Specific antiantigen receptor antibody (anti-mu) induces resting B cells to proliferation but not differentiation. Upon addition of both LPS and anti-mu to cultures, resting B cells again proliferate but do not differentiate. RNA transfer blots of the Ig mRNA 2 days after induction with LPS/anti-mu show a specific deficiency of the 2.4-kilobase (kb) mu secretory mRNA, whereas the levels of the 2.7-kb mu membrane and 1.2-kb kappa light chain mRNAs are as high as in cells treated with LPS alone. Between days 3 and 4 after treatment with both reagents, reductions of mu membrane and, to a smaller extent, kappa mRNA become apparent. As measured by nuclear run-on transcription experiments at day 2, the transcription rates of Ig mu and the Ig kappa transcription units are equal in both induction experiments. Only at later stages do the LPS/anti-mu-treated cells transcribe Ig genes at a lower rate. Thus, the anti-mu treatment, drastically reducing the mu secretory mRNA production at early stages, represents a negative regulation occurring primarily at the posttranscriptional level.
Selective loss of a subset of T helper cells in active multiple sclerosis.
Authors: Rose L M , Ginsberg A H , Rothstein T L , Ledbetter J A , Clark E A
Published: May 2006

Patients with active multiple sclerosis (MS) have a selective loss of a subset of T helper cells (Th), detectable by two-color fluorescence-activated cell sorter analysis of peripheral blood lymphocytes. By using pairs of monoclonal antibodies to the T-cell subset markers CD4 (Th) and CD8 [T suppressor/cytotoxic cell (Ts)] and the common leukocyte markers Lp220 and Lp95-150, five phenotypically distinct T-cell subsets have been identified in peripheral blood: two CD4+ Th cell subsets and three CD8+ Ts cell subsets. The frequencies and absolute numbers of these five populations were measured in patients with active and inactive MS and were compared with those in healthy age-matched controls and in patients with other neurologic diseases. A high frequency of patients with active MS (80%) had a selective reduction of one Th subset (CD4+ Lp220+) compared with normal controls (P less than 0.001) or patients with inactive MS (P less than 0.001). Three patients examined sequentially had a further loss of the Lp220+ Th subset as disease activity progressed. The proportion of two Ts subsets was also abnormal in patients with active MS, but this defect was not restricted to that group. Total Th and Ts cell frequencies and Th/Ts ratios were not significantly different between patient and normal control groups. Thus, two-color analysis of T-cell subsets may be a more sensitive indicator than conventional single-marker assays of abnormal immune status in MS patients.
Enrichment of hematopoietic precursor cells and cloning of multipotential B-lymphocyte precursors.
Authors: McKearn J P , McCubrey J , Fagg B
Published: May 2006

A simple one-step isolation technique significantly enriched mouse fetal liver cells that respond to interleukin 3 (IL-3), a multilineage hematopoietic growth factor. The fetal liver cell subpopulation isolated with monoclonal antibody AA4 contained 50- to 100-fold higher frequencies of multipotential (CFU-mix) or restricted (CFU-G/M, BFU-E) erythroid/myeloid precursors as well as precursors that differentiate to become mature B lymphocytes [CFU-mix = erythroid and myeloid colony-forming unit(s); CFU-G/M = CFU-granulocyte/macrophage; BFU-E = burst-forming unit-erythroid]. The B-lymphocyte precursors could be cloned in single-cell cultures when IL-3-containing supernatants were present. Growth of these clones was supported by purified IL-3 but not by purified IL-2. Stable growth has been maintained for greater than 6 mo in the presence of IL-3. Such clones express on their cell surface low amounts of class I major histocompatibility complex antigens and high amounts of AA4, GF1, and leukocyte common glycoprotein 200 antigens. They lack detectable rearrangements of their Ig-encoding genes [joining region heavy and light (kappa, lambda) chain genes], even after subcloning, but maintain their capacity to differentiate to mature B lymphocytes committed to multiple Ig specificities.
Molecular cloning and the nucleotide sequence of cDNA for neuron-specific enolase messenger RNA of rat brain.
Authors: Sakimura K , Kushiya E , Obinata M , Odani S , Takahashi Y
Published: May 2006

The cDNAs to mRNA for rat gamma gamma enolase (neuron-specific enolase; NSE; EC 4.2.1.11) were isolated from a cDNA library by using differential colony hybridization and a hybrid-selected translation assay. By overlapping of the nucleotide sequences of several cDNA inserts, it was found that they spanned 2232 base pairs (bp) which included 1299 bp of the complete coding region, 68 bp of the 5' noncoding region, and 848 bp of the 3' noncoding region, including a polyadenylylation signal. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was composed of 433 amino acids. Southern blot analysis with a cDNA insert detected one hybridizing fragment in rat genomic DNA digested with several different restriction enzymes. Dot-blot and transfer hybridization analyses of poly(A)+ RNA from developing rat brains showed an increase of NSE mRNA 10-30 days after birth.
Mapping of functional domains in adenovirus E1A proteins.
Authors: Krippl B , Ferguson B , Jones N , Rosenberg M , Westphal H
Published: May 2006

We have modified the E1A gene of human subgroup C adenovirus by introducing deletions in its coding sequence. Various truncated E1A proteins were expressed in Escherichia coli, purified, and microinjected via glass capillaries into Vero cells. We monitored their movement from the cell cytoplasm to the nucleus and their ability to induce expression of H5dl312, an adenovirus E1A deletion mutant. Our results show that the carboxyl terminus of E1A contains sequences essential for rapid and efficient nuclear localization. Essential information for efficient H5dl312 complementation is contained in an internal region, comprising sequences of both exons of the E1A gene. A first exon-encoded region, however, is sufficient to induce low levels of adenovirus gene expression. Information for nuclear localization and for H5dl312 complementation are therefore encoded by distinct domains of the E1A gene. In addition, we determined that the human c-myc product was unable to complement H5dl312.
Protein microcrystal diffraction and the effects of radiation damage with ultra-high-flux synchrotron radiation.
Authors: Hedman B , Hodgson K O , Helliwell J R , Liddington R , Papiz M Z
Published: May 2006

By using ultra-high-flux synchrotron x-radiation from a wiggler source, good Laue diffraction data have been obtained from protein microcrystals of size 30 X 35 X 10 microns3, mounted wet in glass capillaries. At the flux level of 10(13)-10(14) photons per sec/mm2, the radiation damage is still low enough to allow a large survey of reciprocal space for a microcrystal and a complete survey for a normal-sized protein crystal. The development of sources for ultra-high-intensity synchrotron radiation is thus an important improvement in the technique for determination of structure through protein crystallography as well as in other cases where crystal size is often a limiting factor.
Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester.
Authors: Blenis J , Erikson R L
Published: May 2006

Protein kinase capable of phosphorylating 40S ribosomal protein S6 on serine residues has been detected in chicken embryo fibroblasts. This activity appears to be regulated in direct response to expression of pp60v-src in chicken embryo fibroblasts infected with a temperature-sensitive transformation mutant of Rous sarcoma virus. Partially purified S6 kinase was highly specific for S6 in 40S ribosomal subunits. The S6 kinase was not inhibited by calcium or by the heat-stable inhibitor of cAMP-dependent protein kinase, nor was it activated by phosphatidylserine, diacylglycerol, and calcium. Thus, it is distinct from protein kinase C and cAMP-dependent protein kinase, which are capable of phosphorylating S6 in vitro. The tumor-promoter phorbol 12-myristate 13-acetate also stimulated ribosomal protein S6 kinase activity in serum-starved chicken embryo fibroblasts, whereas phorbol, the inactive analog of phorbol 12-myristate 13-acetate, had no effect. S6 kinase activity stimulated by expression of pp60v-src, by phorbol 12-myristate 13-acetate, or by serum growth factors exhibited similar chromatographic properties upon ion-exchange chromatography. These results suggest that a common protein kinase may be activated by three diverse stimuli all involved in regulating cell proliferation.
Detection of neutral amino acid substitutions in proteins.
Authors: Whitney J B , Cobb R R , Popp R A , O'Rourke T W
Published: May 2006

The field of biochemical genetics relies heavily upon the detection by electrophoresis of genetically determined variants of proteins. Most of these variants differ by substitutions that involve charged amino acids. Genetic variants of another large class, ones that involve substitutions among neutral amino acids, are not easily detected and are often ignored. Ampholyte isoelectric focusing in some cases can separate proteins indistinguishable by standard electrophoresis, including genetic variants of mouse hemoglobins that differ only by neutral amino acid substitutions. A revolutionary variation of isoelectric focusing, in which gradients covering a small pH range are fixed into place in a polyacrylamide gel, provides greater resolution of these nearly identical proteins. Mouse hemoglobin tetramers that differ only by the substitution of alanine for glycine in the alpha-globin chains are resolved by several millimeters with the new technique; by comparison, these tetramers are imperfectly resolved on a standard pH 7-9 isoelectric focusing gel. This improved technique of isoelectric focusing was used to identify a variety of previously unreported genetic variants of mouse hemoglobin alpha chains. Immobilized gradients tailored to the requirements of the proteins being analyzed will extend greatly the ranges of protein variations that can be easily recognized for diverse applications, including genetic quality-control analyses and in studies of genetics, mutagenesis, and evolution.
Increased angiotensin II binding affinity in the nucleus tractus solitarius of spontaneously hypertensive rats.
Authors: Plunkett L M , Saavedra J M
Published: May 2006

Angiotensin II (Ang) binding kinetics were determined in discrete brainstem nuclei of 14-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) by a quantitative autoradiographic technique. Tissue sections were incubated with 125I-labeled [sarcosine-1]Ang, and results were analyzed by computerized densitometry and comparison to 125I-labeled standards. A single class of high-affinity binding sites was identified in the nucleus tractus solitarius, the area postrema, and the inferior olivary nuclei of both SHR and WKY rats. Ang binding affinity was significantly greater in the nucleus tractus solitarius of SHR compared to normotensive WKY rats (0.27 +/- 0.06 X 10(9) M-1 in WKY rats vs. 0.59 +/- 0.15 X 10(9) M-1 in SHR), with no apparent changes in the maximum binding capacity of this area. There were no changes in the Ang binding kinetics of the area postrema or the inferior olivary nuclei. Our results suggest that central Ang activity is altered in established hypertension in a brainstem area of SHR associated with peripheral cardiovascular control.
Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.
Authors: Ebina Y , Edery M , Ellis L , Standring D , Beaudoin J , Roth R A , Rutter W J
Published: May 2006

We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growth factor I, displays insulin-stimulated autophosphorylation of the beta subunit, and mediates insulin-stimulated 2-deoxyglucose uptake. We conclude that the human insulin receptor is synthesized, processed normally, and functional in this heterologous cell system.
Regulation of expression of the human interferon gamma gene.
Authors: Hardy K J , Peterlin B M , Atchison R E , Stobo J D
Published: May 2006

DNA fragments isolated from a genomic clone of human gamma interferon (IFN-gamma) as well as IFN-gamma cDNA were used to map potential regulatory regions of the IFN-gamma gene by DNase I-hypersensitivity analyses. In nuclei from the human T-cell line Jurkat, which can be induced to express the IFN-gamma gene, we observed a strongly hypersensitive site in the first intervening sequence that localized to the only intracistronic repeat element in the gene. DNase I mapping of Jurkat cells was compared to that of several other cell types, including B cells, macrophages, and epithelial cells. The presence of strong intronic hypersensitivity was found only in cells capable of expressing the IFN-gamma gene. No hypersensitivity was found in the 3' regions of the gene. Further, no hypersensitivity was observed when purified genomic DNA from Jurkat was analyzed, suggesting that DNA-protein interactions, and not simply DNA sequence alone, were responsible for DNase I hypersensitivity. The sequence AAGTGTAATTTTTTGAGTTTCTTTT, which is directly in the intronic hypersensitive area of IFN-gamma, is 83% homologous to a nearly identical sequence in the 5' flanking region of the interleukin 2 gene. In interleukin 2, the homologous sequence is about 300 base pairs upstream of that gene's promoter in an area of potential regulatory importance.