bluuebunny/crossref_metadata_2025 huggingface dataset:
Myristic acid stimulation of bacterial bioluminescence in "aldehyde" mutants.
Authors: Ulitzur S , Hastings J W
Published: May 2006

The involvement of long chain aldehyde in bacterial luminescence was known both from its being required for light emission in the in vitro reaction with pure luciferase and from its ability to stimulate luminescence in vivo in a certain class of dark "aldehyde" mutants. We have found that the luminescence of some (but not all) of such aldehyde mutants is also stimulated by long chain aliphatic fatty acids, with a marked specificity for myristic (tetradecanoic) acid. This stimulation has been demonstrated in aldehyde mutants of two species of luminous bacteria, Beneckea harveyi and Photobacterium fischeri. The responses, both in intensity and yield, are proportional to the amount of added tetradecanoic acid over a 1000-fold range, down to 10 pmol ml-1. Unsaturated long chain fatty acids are potent inhibitors of the tetradecanoic acid stimulation, but they do not effect the in vivo luminescence of wild-type bacteria.
Biosynthesis in vitro of fucose-containing glycosphingolipids in human neuroblastoma IMR-32 cells.
Authors: Presper K A , Basu M , Basu S
Published: May 2006

Two different glycolipid:fucosyltransferase activities involved in the biosynthesis in vitro of blood group-related glycosphingolipids have been detected in a membrane preparation isolated from a human neuroblastoma-derived clonal cell line, IMR-32. The membrane preparation contains an alpha (1,2)-fucosyltransferase (EC 2.4.1.89) that catalyzed the transfer of vucose from GDP--[14C]fucose to neolactotetraosylceramide or neolactopentaosylceramide to form types H-I and B-I glycolipids, respectively. The second fucosyltransferase catalyzes the transfer of fucose to lactotriaosylceramide [GlcNAc(beta1-3)Gal(beta1-4)Glc-Cer] to form a tetraglycosylceramide intermediate of the novel Lea-type glycolipid. UDP-galactose:lactotriaosylceramide beta-galactosyltransferase (EC 2.4.1.86) had 4 times the activity of UDP-galactose:alpha-galactosyltransferase (EC 2.4.1.87) when tested under similar conditions. alpha-Fucosyltransferase activities and the incorporation of [14C]fucose into glycoproteins and glycolipids were also compared in cells differentiated in the presence of 4 micron BrdUrd and 6-mercaptoguanosine.
Molecular mechanisms for proton transport in membranes.
Authors: Nagle J F , Morowitz H J
Published: May 2006

Likely mechanisms for proton transport through biomembranes are explored. The fundamental structural element is assumed to be continuous chains of hydrogen bonds formed from the protein side groups, and a molecular example is presented. From studies in ice, such chains are predicted to have low impedance and can function as proton wires. In addition, conformational changes in the protein may be linked to the proton conduction. If this possibility is allowed, a simple proton pump can be described that can be reversed into a molecular motor driven by an electrochemical potential across the membrane.
Bioluminescence of the firefly: key steps in the formation of the electronically excited state for model systems.
Authors: Koo J A , Schmidt S P , Schuster G B
Published: May 2006

The chemcial mechanism for formatin of electronically excited-state molecules from the thermal reaction of dimethyldioxetanone was studied. Light production in the presence of certain easily oxidized aromatic hydrocarbons was found not to conform to the classical mechanistic schemes for chemiexcitation. Detailed investigation of the dioxetanone system revealed light formation by the recently discovered, chemically initiated electron-exchange process. This result is extrapolated to bioluminescent systems. In particular, the key high-energy molecule involved in firefly luminescence, which has been identified as a dioxetanone, is postulated to form excited states as a result of intramolecular electron transfer from the phenoxythiazole moiety to the dioxetanone. Subsequent rapid decarboxylation results in direct formation of an excited single state of the emitting amide.
Temperature-sensitive RNA polymerase II mutations in Chinese hamster ovary cells
Authors: Ingles C. James
Published: May 2006

Mutant Chinese hamster ovary cell lines temperature-sensitive (TS) for growth and containing TS mutations in RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) have been isolated. Wild-type cells were treated with the mutagen
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine and a population of cells possessing mutations in RNA polymerase II was initially selected by isolating α-amanitin-resistant clones at 34°. Of 168 such α-amanitin-resistant isolates screened for temperature sensitivity, nine were TS for growth at 39.5°. By examining the behavior of the α-amanitin resistance of these TS cell lines in somatic cell hybrids, the TS mutation in a number of them was shown to be in RNA polymerase II. Hybrid cells obtained by the fusion of the TS and α-amanitin-resistant cells with cells possessing α-amanitin-sensitive polymerase II grew at both 34° and 39.5°; the TS mutations were recessive. At 34° all the hybrids were α-amanitin-resistant and possessed a mixture of α-amanitin-resistant and sensitive polymerase II. At 39.5° the α-amanitin-resistant polymerase II activities in hybrids of four of the TS cell lines were lost; these four lines were α-amanitin-sensitive and possessed only α-amanitin-sensitive polymerase II. Temperature-insensitive revertants of two of these mutants were isolated. Reversion of the TS phenotype for mutants TsAma
R
-1 and TsAma
R
-8 was accompanied by an alteration in the level of α-amanitin resistance of the RNA polymerase II activities in the revertant cells. Together these data provide convincing evidence that TS mutations in RNA polymerase II can be coselected with α-amanitin resistance.
Thyroid and glucocorticoid hormones synergistically control growth hormone mRNA in cultured GH1 cells.
Authors: Shapiro L E , Samuels H H , Yaffe B M
Published: May 2006

We have previously demonstrated that thyroid hormone controls growth hormone synthesis in GH1 cells and that the induction of the growth hormone response by glucocorticoid appears to be highly dependent on thyroid hormone action. Thyroid hormone induces growth hormone synthesis approximately 5- to 20-fold and cortisol increases this response 2- to 6-fold further. Long-term kinetics of the growth hormone response show that, without added thyroid hormone, cortisol can induce a small-growth hormone response after 48 hr of incubation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of proteins synthesized in intact cells demonstrates that the cortisol enhancement of growth hormone synthesis in cells incubated with thyroid hormone is a relatively selective process. Quantitation of growth hormone mRNA levels by cell-free protein synthesis demonstrates that the regulation of growth hormone synthesis by thyroid and glucocorticoid hormones is explained by a synergistic pretranslational control mechanism, presumably at the level of the growth hormone gene.
Affinity purification of cardiac adenylate cyclase: dependence on prior hydrophobic resolution.
Authors: Homcy C , Wrenn S , Haber E
Published: May 2006

The interaction of cardiac adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] with a variety of nucleotide affinity resins was systematically investigated. None of these resins effectively bound the native, detergent-solubilized enzyme. However, after hydrophobic resolution on an uncharged resin consisting of long-chain alkyl groups linked to agarose via ether bonds, 40% of the adenylate cyclase activity biospecifically adsorbed to an ATP affinity resin. Gel filtration without detergent after hydrophobic chromatography demonstrated that the enzyme eluted in the identical position as the native enzyme chromatographed in the presence of detergent. This preparation almost completely biospecifically adsorbed to the same ATP-resin and was not eluted with 5 mM cyclic AMP, pyrophosphate, or GTP. If the GTP-washed immobilized enzyme was subsequently desorbed with ATP, then expected Gpp(NH)p (5'-guanylyliminodiphosphonate) sensitivity persisted. A preliminary purification scheme that resulted in an approximate 5000-fold increase in specific activity is presented. These observations indicate that a membrane-bound enzyme may appear to be intrinsically hydrophobic only by virtue of aggregation with other hydrophobic constituents and that prior separation of hydrophobic chromatography may permit such proteins to be fractionated subsequently by methods conventionally applied to hydrophilic proteins.
On positivity of pseudo-differential operators
Authors: Fefferman C. , Phong D. H.
Published: May 2006

In this paper we obtain new lower bounds for pseudo-differential operators with non-negative symbols, thus providing a sharper form of Gårding's inequality.
Exposure of DNA bases induced by the interaction of DNA and calf thymus DNA helix-destabilizing protein.
Authors: Kohwi-Shigematsu T , Enomoto T , Yamada M A , Nakanishi M , Tsuboi M
Published: May 2006

The reaction of chloroacetaldehyde with adenine bases in DNA to give a fluorescent product was used to study the availability to intermolecular reaction of positions 1 and 6 of adenine in DNA complexes with calf thymus DNA helix-destabilizing protein. No inhibition of this reaction was observed when heat-denatured DNA was complexed with the protein at a protein/DNA weight ratio of 10:1, compared to free DNA. On the contrary, the same reaction was inhibited markedly for denatured DNA in the presence of calf thymus histone HI at protein/DNA weight ratio of 2:1. Furthermore, the exchange rate for hydrogens of amino and imide groups of DNA bases in DNA strands with deuterium in the solvent was totally unaffected upon complexing of DNA with the DNA helix-destabilizing protein as examined by stopped-flow ultraviolet spectroscopy. These results indicate that the DNA helix-destabilizing protein forms a complex with single-stranded DNA, leaving DNA bases uncovered by the protein. The fluorescence intensity of DNA pretreated with chloroacetaldehyde was amplified by nearly 3-fold upon addition of the DNA helix-destabilizing protein. The possibility of "unstacking" of DNA bases induced by the protein is discussed.
Modulation of ornithine decarboxylase activity in Escherichia coli by positive and negative effectors.
Authors: Kyriakidis D A , Heller J S , Canellakis E S
Published: May 2006

Two effectors of ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) have been extracted from an ODC- (speC-) mutant, Escherichia coli MA 255. One of these is an ODC inhibitor (Mr 15,000 +/- 2000) that is labile to trypsin; its activity increases 20-fold in response to increased polyamine levels in the growth medium. It has additional characteristics similar to those of the ODC antizyme of eukaryote cells: it is a noncompetitive inhibitor of ODC; the complex formed between ODC and the ODC inhibitor can be dissociated with salt to provide active ODC and active ODC inhibitor; furthermore, this E. coli ODC inhibitor is inhibitory to eukaryote ODC. A thermostable nondialyzable factor that activates ODC in vitro has also been extracted from MA255; increased polyamine levels in the growth medium caused a 1.6-fold increase in the activity of this ODC activator. Effectors with comparable activities have also been identified in the parent ODC+ (speC+) strain MA197. The fluctuations of the intracellular levels of these two ODC effectors during the growth of E. coli MA255 have been related to the temporal changes of the activity of ODC in the parent ODC+ MA197 strain. The mode of interaction of these three macromolecules, as reflected in the changes of the activity of ODC, appears to be complex. The results suggest that ODC activity may be controlled post-translationally by macromolecules that act as positive and negative effectors and whose levels fluctuate in response to the concentration of the end products of the reaction.
Light regulation of specific mRNA species in
Lemna gibba
L. G-3
Authors: Tobin Elaine M.
Published: May 2006

Polyadenylated RNA was isolated from
Lemna gibba
L. G-3 and translated in a cell-free system from wheat germ. When plants were placed into complete darkness for 4 days, then returned to light for 18 hr, increased amounts of polyadenylated mRNA for at least two polypeptides were detected by
in vitro
translation over those amounts present in the dark. These two polypeptides have molecular weights of 32,000 and 20,000. The 20,000 dalton polypeptide was identified by immunoprecipitation as the precursor to the small subunit of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. The polyadenylated mRNA that codes for the small subunit is not detectable by immunoprecipitation of translation products in the dark-treated tissue. Plants growing in the light actively synthesize both subunits of ribulose-1,5-bisphosphate carboxylase, but synthesis of these proteins was found to be greatly diminished in plants placed in darkness for 4 days. These results indicate that white light can dramatically affect the steady-state levels of specific polyadenylated mRNAs.
Inducers of DNA synthesis present during mitosis of mammalian cells lacking G1 and G2 phases.
Authors: Rao P N , Wilson B A , Sunkara P S
Published: May 2006

The cell cycle analysis of Chinese hamster lung fibroblast V79-8 line by the premature chromosome condensation method has confirmed the absence of measurable G1 and G2 periods. Sendai virus-mediated fusion of mitotic V79-8 cells with G1 phase HeLa cells resulted in the induction of both DNA synthesis and premature chromosome condensation in the latter, indicating the presence of the inducers of DNA synthesis above the critical level not only throughout S phase, as it is in HeLa, but also during mitosis of V79-8 cells. No initiation of DNA synthesis was observed when G1 phase HeLa cells were fused with mitotic CHO cells. These results indicate that the presence or absence of a G1 period in the cell cycle depends on the levels of the inducers of DNA synthesis present in the cell during mitosis.
Biological significance of alloreactivity: T cells stimulated by Sendai virus-coated syngeneic cells specifically lyse allogeneic target cells.
Authors: Finberg R , Burakoff S J , Cantor H , Benacerraf B
Published: May 2006

In vitro stimulation of spleen cells from mice immune to Sendai virus results in the generation of effector cells that lyse unmodified allogeneic target cells in addition to syngeneic cells modified by virus. These cells are immunologically specific because their lysis may be blocked by cold targets syngeneic to either the stimulator or the responder. These results support our proposal that the development of alloreactivity can be explained by the crossreactivity between modified self major histocompatibility complex antigens and alloantigens. We propose that exposure to autologous major histocompatibility complex antigens modified by foreign antigens in our environment results in the expansion of the pool of T cells that will respond to alloantigens sharing crossreactive determinants.
Corticotropin, lipotropin, and beta-endorphin production by a human nonpituitary tumor in culture: evidence for a common precursor.
Authors: Bertagna X Y , Nicholson W E , Sorenson G D , Pettengill O S , Mount C D , Orth D N
Published: May 2006

A continuous line (DMS-79) of human pulmonary small cell carcinoma cells was shown to secrete immunoreactive adrenocorticotropin (ACTH), lipotropin, and beta-endorphin concomitantly into the culture medium. Gel filtration of the culture medium demonstrated at least five components: high molecular weight material(s) that had ACTH, lipotropin, and beta-endorphin immunoreactivities and materials similar to ACTH, beta-lipotropin, gamma-lipotropin, and beta-endorphin in their immunoreactivities and apparent molecular weights. The same components were observed when gel filtration was carried out in 6 M guanidine-HCl, and the high molecular weight material(s) appeared to consist of more than one component, with molecular weights in the range of 15,000-40,000. Immune affinity chromatography of the high molecular weight component(s) from gel filtration with a specific anti-(1-24)ACTH serum demonstrated that the ACTH, lipotropin, and beta-endorphin immunoreactivities were possessed by the same molecule(s), suggesting that ACTH, lipotropins, and beta-endorphin were derived from a common, high molecular weight precursor.
Reductant-dependent electron distribution among redox sites of laccase.
Authors: Farver O , Goldberg M , Wherland S , Pecht I
Published: May 2006

Rhus laccase (monophenol monooxygenase, monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) an O2/H2O oxidoreductase containing four copper ions bound to three redox sites (type 1, type 2, and type 3 Cu pair), was titrated anaerobically with several reductants having various chemical and thermodynamic properties. The distribution of electron equivalents among the redox sites was found to be reductant dependent. When the data for titration by various reductants of the type 3 site were plotted against those of the type 1 site according to the Nernst formalism, the slope n varied from 2.0 to 1.0. The redox potential of the reductant's first oxidation step is qualitatively correlated with the value of n and is suggested as the factor that modulates the electron distribution. Such a behavior implies a nonequilibrium situation. A very good simulation of the data was provided by an analysis assuming a formally variable cooperativity between the two type 3 copper ions. This apparent variability is suggested to result from a process whereby sufficiently strong reductants induce a transition of the type 3 site from a cooperative two-electron acceptor to a pair of independent one-electron acceptors. This uncoupled state of the type 3 site is considered metastable. Other possible models were also investigated. Summarizing the available data, we conclude that the two-electron accepting behavior of the 330-nm chromophore is the exception rather than the rule.
A photo-CIDNP study of the interaction of oligonucleotides with gene-5 protein of bacteriophage M13.
Authors: Garssen G J , Kaptein R , Schoenmakers J G , Hilbers C W
Published: May 2006

It is shown that photo-CIDNP effects (CIDNP, chemically induced dynamic nuclear polarization) can be generated in the 360-MHz proton NMR spectrum of gene-5 protein from bacteriophage M13. This technique is used to determine the number of tyrosyl residues at the surface of the protein and to assign the resonances from the 3,5-ring protons of these residues. The DNA-binding site of the protein is investigated by formation of complexes with oligonucleotides. Complex formation leads to shifting and/or quenching of the photo-CIDNP emission signals of the surface tyrosines, implying that they are involved in DNA-protein interaction. These experiments are complemented by studying the complex formation of Lys-Tyr-Lys to poly(A).
Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.
Authors: Buening M K , Wislocki P G , Levin W , Yagi H , Thakker D R , Akagi H , Koreeda M , Jerina D M , Conney A H
Published: May 2006

The tumorigenicities of benzo[a]pyrene and each optical enantiomer of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides derived from trans-7,8-dihydroxy-7,8-dihydrobenzol[a]pyrene were tested by sequential intraperitoneal injection of mice with 1,2, and 4 nmol, or with 2, 4, and 8 nmol of each compound on the 1st, 8th, and 15th day of life, respectively. The experiment was terminated when the animals were 34--37 weeks old. (+)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2] had exceptional tumorigenicity, whereas benzo[a]-pyrene and the other three optically pure isomers of the benzo[a]pyrene 7,8-diol,9,10-epoxides had little or no activity. These results demonstrate differences in the carcinogenic activities of optically active isomers of a polycyclic hydrocarbon diol epoxide. Eleven percent of control mice had pulmonary tumors, whereas 71% and 100% of the mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2, respectively, had pulmonary tumors. Control mice had an average of 0.12 pulmonary tumors per mouse, whereas mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2 had 1.72 and 7.67 pulmonary tumors per mouse, respectively. Mice treated with 14 nmol of (-)-BP-7alpha,8beta-diol-9beta,10beta-epoxide 2, (-)-BP-7beta,8alpha-diol-9beta,10beta-epoxide 1, or (+)-BP-7alpha,8beta-diol-9alpha,10alpha-epoxide 1 had 0.13, 0.25, and 0.34 pulmonary tumors per animal, respectively.
Resolution of complex III of the mitochondrial electron transfer chain into two component complexes.
Authors: Fry M , Green D E
Published: May 2006

Complex III can be resolved into an electron transfer complex and an ion transfer complex. The electron transfer complex contains exclusively the oxidation-reduction proteins of complex III, and the ion transfer complex the capability for augmenting cation-induced fluorescence of 8-anilino-1-naphthalenesulfonic acid. This finding has important implications for the molecular mechanism of mitochondrial energy coupling.
Construction and characterization of a 2.5-kilobase procollagen clone.
Authors: Lehrach H , Frischauf A M , Hanahan D , Wozney J , Fuller F , Crkvenjakov R , Boedtker H , Doty P
Published: May 2006

Recombinant bacterial plasmids have been constructed by inserting double-stranded chicken procollagen cDNA sequences linked to chemically synthesized decanucleotides containing HindIII sites into the HindIII site of pBR322. After transformation of Escherichia coli chi1776, colonies were selected by ampicillin resistance and recombinants containing procollagen sequences were identified by colony hybridization to 32P-labeled procollagen cDNA. The inserts from three recombinant plasmids, pCg10, pCg13, and pCg45, were 1200, 2200, and 2550 base pairs long respectively. Their sequence homology has been established by restriction mapping and crosshybridization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts, pCg45 has been positively identified as containing the pro alpha2 collagen sequence by partial determination of the DNA sequence of its ends: it has a short thymine-rich sequence at one end and a sequence coding for residues 478--499 in the chicken alpha2 chain at the other end.
Ca2+-dependent protein phosphorylation system in membranes from various tissues, and its activation by "calcium-dependent regulator".
Authors: Schulman H , Greengard P
Published: May 2006

Analysis of membranes from a variety of tissues has revealed a widespread distribution of a protein phosphorylation system dependent on the presence of both Ca2+ and "calcium-dependent regulator" (CDR). This protein phosphorylation system has been studied in some detail in nervous tissue. Neuronal membranes contain a protein phosphorylation system that requires Ca2+ and a soluble heat-stable protein [Schulman, H. & Greengard, P. (1978) Nature (London) 271, 478--479]. This protein has been purified to homogeneity from bovine cerebral cortex, with use of an assay based on its ability to stimulate Ca2+-dependent protein phosphorylation in membranes. This protein kinase activator appears to be identical to CDR of cyclic nucleotide phosphodiesterase. Throughout its purification, this single entity was found to activate both Ca2+-dependent protein kinase and cyclic nucleotide phosphodiesterase. The kinase activator purified here and authentic CDR were equally effective in their ability to activate Ca2+-dependent protein kinase.
Structure of the lambda att sites generated by int-dependent deletions.
Authors: Hoess R H , Landy A
Published: May 2006

Bacteriophage lambda integrates into the chromosome of its Escherichia coli host by means of a site-specific recombination between a locus on the phage chromosome (phage att site) and a locus on the bacterial chromosome (bacterial att site). The nucleotide sequence of four lambda att sites altered in site-specific recombination has been determined. The int-dependent deletions that generated these att sites have one end point within the phage att site and extend either to the left or to the right. As a result of the new internucleotide bond created by deletion formation, these phage have alterations in the 15-base-pair common core region. The new DNA sequences brought to the att sites by the deletions, designated delta for regions to the left and delta' for regions to the right, do not share any discernible homology with their analogous counterparts in the phage att site arms, P and P', respectively, or with the bacterial att site arms, B and B', respectively. The finding of alterations in the 15-base-pair common core region necessitates a reinterpretation of the genetic properties of these att sites in site-specific recombination. The structure of these sites in relation to their genetic properties can be viewed as being consistent with a model in which the only specificity elements in int-dependent site-specific recombination are the common core region, O, and the phage arms, P and P'.
Identification of a membrane protein as a histidine transport component in Salmonella typhimurium.
Authors: Ames G F , Nikaido K
Published: May 2006

A component of high-affinity histidine transport in Salmonella typhimurium has been identified. It is a basic (pI about 9.0) membrane-bound protein, the P protein. It is shown to be coded for by the distal half of the previously described hisP gene by analysis of numerous hisP mutants, two of which exhibit P proteins with altered electrophoretic mobilities. Upon separation of the cytoplasmic (inner) from the outer membrane, it can be shown that the P protein is located in the cytoplasmic membrane. The P protein is under the same regulatory controls as histidine transport--i.e., transport operon promoter dhuA and nitrogen regulation. A wild-type cell contains about 200 molecules of P protein. As a result of this work we now divide the hisP gene into two genes: the hisP gene proper and the hisQ gene, which codes for another essential component of histidine transport, the Q protein. The P protein was shown previously by genetic analysis to interact with the periplasmic histidine-binding protein J, another essential component of histidine transport. Possible mechanism for the interaction of the J, P, and Q components in histidine transport, and of P and Q in lysine/arginine/ornithine transport, are discussed.
Identification of four trans-3,4-dihydrodiol metabolites of 7,12-dimethylbenz[a]anthracene and their in vitro DNA-binding activities upon further metabolism.
Authors: Chou M W , Yang S K
Published: May 2006

Trans-3,4-dihydrodiols of 7,12-dimethylbenz[a]anthracene (7,12-Me2BA), 7-methyl-12-hydroxymethylbenz[a]anthracene (7-Me-12-OHMeBA), 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHMe-12-MeBA), and 7,12-di(hydroxymethyl)benz[a]anthracene [7,12-(OHMe)2BA] have been identified as metabolites of the potent carcinogenic and adrenocorticolytic agent 7,12-MeBA. The four trans-3,4-dihydrodiols were identified by their (i) ultraviolet-visible absorption and fluorescence properties, (ii) different retention times on both reversed-phase and normal-phase high-pressure liquid chromatography, (iii) mass spectral analysis, and (iv) inability to form vicinal cis-acetonides. Upon further metabolism by liver microsomes, the trans-3,4-dihydrodiols of 7,12-Me2BA, 7-Me-12OHMeBA, and 7-OHMe-12-MeBA were found to give rise to products that bind more strongly to DNA in vitro than do the products of 7,12-Me2BA. The evidence suggests that one or more of the four trans-3,4-dihydrodiols may be the proximate carcinogenic and adrenocorticolytic metabolites.
Long-term growth and differentiation of Xenopus oocytes in a defined medium.
Authors: Wallace R A , Misulovin Z
Published: May 2006

Xenpus laevis oocytes over a size range of 0.15--0.78 mm3 were dissected from their follicles and cultured in a defined medium for up to 28 days. Oocytes grew at average rates of 0.021 mm3.day-1 in the absence of insulin and 0.030 mm3.day-1 in the presence of insulin. The latter average growth rate corresponds to the fastest growth rate reported to date for oocytes in vivo. Oocytes grown in vitro can reach a size of at least 1.43 mm3, which is larger than the maximum size generally found in vivo. During growth in vitro; oocytes also acquire both a normal pigment pattern and, once they reach about 0.7 mm3, the ability to undergo complete maturation as a response to externally applied progesterone. These results show that Xenopus oocytes freed of their follicular investments are able to grow and differentiate in vitro.
Complex population of mRNA sequences in large polyadenylylated nuclear RNA molecules.
Authors: Hahn W E , Van Ness J , Maxwell I H
Published: May 2006

Polyadenylylated heterogeneous nuclear RNA [poly(A)-hnRNA] from mouse brain was subjected to electrophoresis in agarose gels containing CH3HgOH, and molecules larger than 8 kilobases or 13 kilobases were recovered. cDNA was then transcribed from polyadenylylated RNA fragments cleaved from these large molecules. The resulting cDNA hybridized almost to completion with poly(A)-mRNA isolated from mouse brain polysomes. From the hybridization kinetics of this cDNA with its template RNA, it was estimated that the sequence complexity of the 3'-proximal sequences (of the same average size as mRNA) of the greater than 8 kilobase poly(A)-hnRNA was about 57,000 kilobases. The sequence complexity of poly(A)-mRNA, estimated from the template-driven hybridization kinetics of its respective cDNA, was about 110,000 kilobases. It is concluded that most, if not all, of the 3'-proximal sequences of large poly(A)-hnRNA molecules are homologous with mRNA in the mouse brain and that at least 40,000 different mRNA sequences (or portions of mRNA sequences) are represented in the 3'-proximal sequences of greater than 8 kilobase poly(A)-hnRNA.
Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line.
Authors: Nunberg J H , Kaufman R J , Schimke R T , Urlaub G , Chasin L A
Published: May 2006

Methotrexate-resistant Chinese hamster ovary cells selected for high resistance by progressive increments of methotrexate in the culture medium have levels of dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) 200 times that of sensitive cells and a corresponding increase in the number of copies of the dihydrofolate reductase gene. The resistant cells contain an expanded region on the second chromosome (homogeneously staining region) that is not present in sensitive cells. In situ hybridization of DNA complementary to dihydrofolate reductase mRNA shows that the dihydrofolate reductase genes are specifically localized to the homogeneously staining region of this chromosome in the resistant cells.
Ecdysone-inducible functions of larval fat bodies in
Drosophila
Authors: Lepesant Jean-Antoine , Kejzlarova-Lepesant Jana , Garen Alan
Published: May 2006

Late in the third instar larval stage of
Drosophila melanogaster
, the titer of the steroid hormone ecdysone increases sharply. This increase is blocked in the temperature-sensitive mutant
ecd
1
after a temperature shift from 20°C to 29°C. The mutant was used to prepare three samples of late third instar larvae with different titers of ecdysone; the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysone-supplemented food, and also high in a third sample that was kept at 20°C, providing a control for normal development. The effect of the high titer of ecdysone on proteins of the larval fat bodies was examined by comparing two-dimensional gel electrophoresis patterns of total proteins in stained gels. There were proteins at five positions in the gels for the high-ecdysone samples that were not detected at the corresponding positions in the gel for the low-ecdysone sample. The effect of ecdysone on these proteins was further studied by injecting [
35
S]methionine into the larvae at both early and late third instar stages, in order to label proteins synthesized before and after the increase in ecdysone titer. The results indicate that ecdysone induces two major responses in the fat bodies; certain proteins that were synthesized earlier in the fat bodies and secreted into the hemolymph are incorporated back into the fat bodies, and other proteins are newly synthesized. Attempts to induce prematurely the synthesis of the new proteins by exposing early third instar larvae to exogenous ecdysone were unsuccessful, suggesting that development must proceed further before the fat bodies can respond to ecdysone.

By
in vitro
translation of RNA isolated from fat bodies of low-and high-ecdysone samples of larvae, it was shown that ecdysone greatly increases the amount of translatable messenger RNA for one of the newly synthesized proteins. A clone of DNA complementary to the induced messenger RNA has been isolated from a population of λ bacteriophage carrying segments of the
Drosophila
genome. Using the cloned DNA to measure amounts of complementary poly(A)-RNA in the fat bodies by DNA·RNA hybridization, we detected about 50 times more complementary poly(A)-RNA in the high-ecdysone sample of larvae than in the low-ecdysone sample. This finding provides direct evidence that ecdysone induces an increase in the amount of the messenger RNA. The ecdysone-induced appearance of a major messenger RNA in late third instar larval fat bodies represents a developmental response to ecdysone that appears to be gene-specific, tissue-specific, and stage-specific, and it has exceptionally favorable features for further molecular studies of the control of gene expression by a steroid hormone.
Structural analysis of the ADHS electromorph of Drosophila melanogaster.
Authors: Fletcher T S , Ayala F J , Thatcher D R , Chambers G K
Published: May 2006

Population geneticists have often determined the fitness differences that account for the dynamics of naturally occurring genetic polymorphisms. However, to understand causal aspects of evolutionary processes requires, in addition, investigation of the physiological and molecular structural differences underlying adaptively significant genetic polymorphisms. The characteristics of the alcohol dehydrogenase gene--enzyme system in Drosophila melanogaster make it well suited for this kind of study. Natural populations of this species are polymorphic for two electrophoretically detectable variants, ADHF and ADHS, of the enzyme. Structural studies reported here reveal that the two variants differ by (at least) a single amino acid replacement, threonine in ADHF for lysine in ADHS.
Genetics of the large, external, transformation-sensitive (LETS) protein: assignment of a gene coding for expression of LETS to human chromosome 8.
Authors: Owerbach D , Doyle D , Shows T B
Published: May 2006

Techniques have been developed to analyze the genetics of the large, external, transformation-sensitive (LETS) protein (fibronectin). External membrane proteins of human-mouse somatic cell hybrids with reduced numbers of human but not mouse chromosomes were labeled by lactoperoxidase-catalyzed iodination. Cell surface proteins were identified after sodium dodecyl sulfate/polyacrylamide gel electrophoresis by autoradiography of the dried gel. The LETS protein was identified in parental human cells, and LETS segregated in human-mouse cell hybrids formed from human WI-38 fibroblasts and a mouse L-cell line not expressing LETS. The LETS protein segregated concordantly with the chromosome 8 enzyme marker glutathione reductase (EC 1.6.4.2) and human chromosome 8. These findings demonstrate that a gene, LETS, encoded on chromosome 8, is responsible for the LETS protein expression in humans. Because LETS has been implicated in tumorigenicity and cellular transformation, it is of interest that rearrangement or modifications in the number of chromosome 8 have been associated with certain forms of cancer.
Generation of b-wave currents in the skate retina.
Authors: Kline R P , Ripps H , Dowling J E
Published: May 2006

Potassium kinetics within the skate retina were monitored extracellularly with K+-selective electrodes. Two sources of K+ efflux were detected in response to photic stimulation: one in the distal retina in the region of the outer plexiform layer, and the other at a more proximal location near the border between the inner nuclear and inner plexiform layers. The magnitude of the K+ efflux at these retinal depths was affected differently by spot and full-field illumination, suggesting that the two sources originate from different classes of neuron. There is evidence that both sources are associated with current sinks provided by the Müller cells, thereby establishing radial current paths along the lengths of these elements. We have proposed a model in which asymmetries in the magnitudes of these currents give rise to the b-wave of the electroretinogram. Extracellular field potentials recorded differentially at various retinal depths, and in response to changes in stimulus configuration, were consistent with predictions of the model.
Cell-free synthesis and processing of the proteins of poliovirus.
Authors: Shih D S , Shih C T , Kew O , Pallansch M , Rueckert R , Kaesberg P
Published: May 2006

Poliovirus RNA can be translated completely and accurately in rabbit reticulocyte lysates; the nascent poly-protein is processed to give primary products 1a, X, and 1b indistinguishable from those made in poliovirus-infected HeLa cells. The capsid precursor protein 1a is processed to form the capsid proteins VP0, VP1, and VP3, while the noncapsid precursor 1b is processed to form protein 2.
Interaction of bacteriophage lambda repressor with nonoperator DNA containing single-strand gaps.
Authors: Sussman R , Resnick J , Calame K , Baluch J
Published: May 2006

In direct binding assays, purified lambdaind+ repressor displayed high affinity for nonoperator DNA containing single-strand gaps. Its affinity for this same DNA but completely double-stranded, nicked, or denatured was considerably lower. In contrast, purified lambdaind- repressor had 1/10th the affinity for the gapped DNA, a level comparable to that of purified lac repressor. In the presence of limiting amounts of ind+ repressor, nonoperator DNA containing gaps could be shown to compete effectively with lambda DNA for binding of repressor. A previous model of lambda induction [Sussman, R. & Ben-Zeev, H. (1975) Proc. Natl. Acad. Sci. USA 72, 1973--1976], based on the assumption that this phenomenon involves the binding of repressor to lesions in the host DNA, is reevaluated in the light of the data reported here.
Change in quantity and size distribution of small circular DNAs during development of chicken bursa.
Authors: DeLap R J , Rush M G
Published: May 2006

Small circular DNAs ranging in contour length from 0.06 to 3.5 micrometers have been isolated from bursas of 19-day chicken embryos and 4- to 5-week-old chickens. Small circular DNA is present in bursas of 19-day embryos at approximately 0.2 molecules per cell and is very heterogeneous, lacking distinct size classes; most molecules have contour lengths of less than 0.04 micrometers. In contrast, small circular DNA is present in bursas of 4- to 5-week old chickens at about 4 molecules per cell, and although this DNA is still heterogeneous, it contains a major distinct class of molecules 0.8 micrometers in size. These small circular DNAs may be products of developmental gene rearrangements occurring in the chromosomal DNA of lymphocytes in the bursa.
Structure of the oxygen adduct intermediate in the bacterial luciferase reaction:
13
C nuclear magnetic resonance determination
Authors: Ghisla Sandro , Hastings J. Woodland , Favaudon Vincent , Lhoste Jean-Marc
Published: May 2006

By using FMN enriched in
13
C (90%) at position C-4a, we have conclusively shown that the reaction of molecular oxygen with bacterial luciferase-bound FMNH
2
forms an adduct at the 4a position. Consistent with this are
13
C NMR studies of FMN and other flavin compounds which show that this carbon should be unusually reactive in the reduced 1,5-dihydroflavins with respect to electrophilic attacks.
Bovine protein C: amino acid sequence of the light chain.
Authors: Fernlund P , Stenflo J , Tufvesson A
Published: May 2006

The amino acid sequence of the light chain of bovine protein C was determined by sequenator analysis of the carboxymethylated light chain and fragments obtained by cyanogen bromide treatment, tryptic digestion after blocking of lysine residues, and cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole). The sequence was (in the standard one-letter code) A-N-S-F-L-X-X-L-R-P-G-N-V-X-R-X-C-S-X-X-V-C-X-F-X-X-A-R-X-I-F-Q-N-T-X-D-T-M-A-F-W-S-K-Y-S-D-G-D-Q-C-E-D-R-P-S-G-S-P-C-D-L-P-C-C-G-R-G-K-C-I-H-G-L-G-G-F-R-C-D-C-A-E-G-W-E-G-R-F-C-L-H-E-V-R-F-S-N-C-S-A-E-B-G-G-C-A-H-Y-C-M-E-E-E-G-R-R-H-C-S-C-A-P-G-Y-R-L-E-D-D-H-Q-L-C-V-S-K-V-T-F-P-C-G-R-L-G-K-R-M-E-K-K-R-K-T-L. The first eleven glutamic acid residues were carboxylated to gamma-carboxyglutamic acid (X). The NH2-terminal, vitamin K-dependent part showed an extensive homology to both prothrombin and factor X, whereas the rest of the chain showed a strong homology to factor X but little similarity to prothrombin.
Translation of polyoma virus T antigens in vitro.
Authors: Hunter T , Hutchinson M A , Eckhart W
Published: May 2006

Polyoma virus-specific RNA isolated from the cytoplasm of lytically infected cells can be translated in vitro to yield three T antigens, of Mrs approximately 90,000, 60,000, and 22,000. The tryptic peptide patterns of the T antigens synthesized in vitro are similar or identical to the patterns of the corresponding proteins in polyoma-infected cells. All three proteins incorporate methionine donated from initiator tRNA in vitro. Polyoma cRNA codes for a protein that is slightly larger than the 22,000 T antigen and that, by other criteria, is similar to the 22,000 T antigen. Translation of cRNA does not yield the 90,000 and 60,000 T antigens, suggesting that the generation of the mRNAs for these T antigens requires the removal of intervening sequences. The mRNA for the 90,000 T antigen is smaller than the mRNAs for the 22,000 and 60,000 proteins. All three proteins share common NH2-terminal sequences, and the 60,000 T antigen may be translated partially in a different reading frame from sequences also coding for the 90,000 T antigen. The demonstration that polyoma virus codes for three different T antigens raises the possibility that all three proteins may be involved in cell transformation.
Chicken ovalbumin is synthesized and secreted by Escherichia coli.
Authors: Fraser T H , Bruce B J
Published: May 2006

By recombinant DNA methods, the chicken ovalbumin structural gene has been fused to Escherichia coli lac transcriptional and translational control regions. When a plasmid containing the hybrid gene was introduced into E. coli, a protein identified as ovalbumin by immunoreactivity and sodium dodecyl sulfate/polyacrylamide gel electrophoresis was synthesized. The chicken ovalbumin made in bacteria was full length (43,000 daltons) and constituted 1.5% of the cellular protein. In addition, the microbially synthesized ovalbumin was secreted through the cell membrane into the periplasmic space of E. coli. The ability of the E. coli secretory apparatus to recognize chicken ovalbumin, which is normally synthesized and secreted in hen oviducts, suggests that common features exist in the secretion-recognition mechanisms found in these two organisms. The bacterial synthesis of significant amounts of chicken ovalbumin demonstrates that the E. coli cellular machinery may be utilized to synthesize a higher eukaryotic protein which is relatively stable in the bacterial intracellular environment.
Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase
Authors: Chua Nam-Hai , Schmidt Gregory W.
Published: May 2006

A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] has been identified among the products of cell-free translation of polyadenylated RNA from spinach and pea. In both cases, the precursor is larger than the mature protein by 4000-5000 daltons. Upon incubation of post-ribosomal supernatants of the
in vitro
protein synthesis mixtures with purified intact chloroplasts, the pea and spinach precursors are transported interchangeably into the chloroplasts and processed to the mature size and charge. Moreover, the newly transported small subunits are found to assemble with endogenous large subunits to form the holoenzyme. In contrast, a precursor to the
Chlamydomonas reinhardtii
small subunit is not taken up by higher plant chloroplasts, indicating the specificity of the transport events. Together, these results demonstrate that the
in vitro
reconstruction of the post-translational transport of the higher plant precursors is physiologically significant.
Mechanism of dexamethasone inhibition of plasminogen activator in rat hepatoma cells.
Authors: Seifert S C , Gelehrter T D
Published: May 2006

Glucocorticoids rapidly and completely inhibit intracellular plasminogen activator activity in rat hepatoma cells, as assayed by the solubilization of 125I-labeled fibrin. Experiments in which extracts of dexamethasone-treated cells are mixed with extracts of control cells demonstrate an inhibitor of protease activity. Plasminogen activator is found primarily in the particulate fraction of cell lysates, whereas the inhibitor is localized in the soluble fraction. Variant cells have been isolated previously that are fully resistant to the dexamethoasone inhibition of plasminogen activator activity. These variants have no demonstrable inhibitor activity, whereas plasminogen activator in these cells is fully sensitive to inhibitor from wild-type cells. Thus, the basis for hormone resistance appears to be the failure of dexamethasone to induce an inhibitor.
Marker rescue and partial replication of bacteriophage T7 DNA.
Authors: Burck K B , Miller R C
Published: May 2006

Experiments reported here show that some UV-irradiated wild-type T7 phage markers can be rescued efficiently by coinfection with T7 amber mutant phage in a permissive host. Other results show that the segments of a UV-irradiated genome that replicate efficiently are those that also are rescued efficiently during a marker rescue experiment. At higher doses, fewer markers are rescued efficiently and fewer segments of the genome replicate efficiently. The results clearly indicate that the probability of marker rescue is correlated with the ability of the DNA containing the marker to replicate. Sucrose density gradient analysis shows that UV irradiation does not produce double-strand scissions in T7 DNA at doses used here. Therefore, the partial replication and rescue of markers from the left end of the genome is not due simply to injection of only the left end of the T7 DNA.