bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.
Authors: Himawan J S , Richardson C C
Published: May 2006

Gene 5 protein of bacteriophage T7 is a nonprocessive DNA polymerase. During infection of Escherichia coli, T7 annexes the host protein thioredoxin for use as a processivity factor for T7 DNA polymerase. We describe here a genetic method to investigate the interaction between T7 gene 5 protein and E. coli thioredoxin. The strategy is to use thioredoxin mutants that are unable to support the growth of wild-type T7 phage to select for T7 revertant phage that suppress the defect in thioredoxin. A thioredoxin mutation that replaces glycine at position 74 with aspartic acid fails to support the growth of wild-type T7. This mutation is suppressed by six different mutations within T7 gene 5, each of which results in a single amino acid substitution within gene 5 protein. Three of the suppressor mutations are located within the putative polymerization domain of gene 5 protein, and three are located within the putative 3'-to-5' exonucleolytic domain. Each suppressor mutation alone is necessary and sufficient to confer the revertant phenotype.
In search of a physical theory of time.
Authors: Hsü K J
Published: May 2006

The variable time considered in science is a measurement. The relation of time to other variables is expressed in rates, and the variable time is seldom explicitly included in equations describing chemical reactions or radioactive decays. Recalling that the Greek word atom is applicable to indivisible particles in both spatial and temporal worlds, the term chronon is proposed to designate elementary particles of time in parallel to the atom in space. Considerations of interactions of chronons with material and nonmaterial objects could promote understanding of several paradoxes in physical and biological sciences.
Quadruplex structure of d(G3T4G3) stabilized by K+ or Na+ is an asymmetric hairpin dimer.
Authors: Scaria P V , Shire S J , Shafer R H
Published: May 2006

The ends of chromosomes contain repeats of guanine-rich sequences that can assume highly compact conformations and are presumed necessary for their biological role in chromosomal stabilization and association. We have investigated the conformational behavior of d(G3T4G3) as a function of the addition of either KCl or NaCl, in the concentration range of 50-200 mM, by using a spectrum of physical techniques and conclude that these salts induce a quadruplex species composed of two strands, each in a hairpin conformation. When salt is added, a large positive signal appears near 290 nm in the CD spectra. UV thermal denaturation curves show a single concentration-dependent transition and provide data for quantitating the thermodynamics of quadruplex formation. In electrophoresis experiments, the quadruplex structure migrates as a single species and more rapidly than the unstructured single strand. NMR spectra in the presence of KCl or NaCl indicate that the structure formed is asymmetric. Equilibrium ultracentrifugation studies confirm that these quadruplexes are composed of two strands of d(G3T4G3). Possible models for this structure are discussed.
Banach-Tarski paradox using pieces with the property of Baire.
Authors: Dougherty R , Foreman M
Published: May 2006

In 1924 Banach and Tarski, using ideas of Hausdorff, proved that there is a partition of the unit sphere S2 into sets A1,...,Ak,B1,..., Bl and a collection of isometries [sigma1,..., sigmak, rho1,..., rhol] so that [sigma1A1,..., sigmakAk] and [rho1B1,..., rholBl] both are partitions of S2. The sets in these partitions are constructed by using the axiom of choice and cannot all be Lebesgue measurable. In this note we solve a problem of Marczewski from 1930 by showing that there is a partition of S2 into sets A1,..., Ak, B1,..., Bl with a different strong regularity property, the Property of Baire. We also prove a version of the Banach-Tarski paradox that involves only open sets and does not use the axiom of choice.
Ligand-induced phosphorylation of the murine interleukin 3 receptor signals its cleavage.
Authors: Mui A L , Kay R J , Humphries R K , Krystal G
Published: May 2006

The murine interleukin 3 receptor (mIL-3R) is a heterodimer consisting of a 70-kDa alpha subunit and one of two alternative 120-kDa beta subunits termed beta IL-3 and beta c. beta IL-3 (originally called Aic2A) is capable of binding mIL-3 by itself, whereas beta c (Aic2B) does not bind any ligand on its own but increases the affinity of mIL-3, murine granulocyte/macrophage-colony-stimulating factor, and mIL-5 for their respective alpha subunits. Interestingly, although the mIL-3R does not possess tyrosine kinase activity, its beta IL-3 subunit does become tyrosine phosphorylated upon binding mIL-3. To further investigate the properties of this subunit, we have purified it from the cell line B6SUtA1, which expresses a high level of mIL-3R. Intriguingly, studies comparing the stability of the 140-kDa, tyrosine-phosphorylated form of this subunit with its 120-kDa, non-tyrosine-phosphorylated form reveal that the former is far less stable and is rapidly degraded to a 70-kDa fragment. Mixing experiments demonstrate that the differential stability of the two forms is due to an intrinsic difference in protease susceptibility. Phosphatase studies indicate that the higher protease susceptibility of the tyrosine-phosphorylated beta IL-3 is due to the presence of both phosphotyrosine and phosphoserine residues. Western analyses using an anti-N-terminal mIL-3R beta IL-3 chain antibody reveal that this proteolytic cleavage also occurs rapidly in intact cells following stimulation with mIL-3 and occurs at the cell surface, since it takes place within minutes at 37 degrees C, is observed with purified plasma membranes, and is not inhibited by chloroquine. This degradative step may play an important role in the mechanism of action of mIL-3.
Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.
Authors: Hellmich M R , Pant H C , Wada E , Battey J F
Published: May 2006

Recent studies have shown that there exists a family of protein kinases structurally and functionally related to the yeast cell cycle regulatory kinase cdc2 [Meyerson, M., Faha, B., Su, L.-K., Harlow, E. & Tsai, L.-H. (1991) Cold Spring Harbor Symp. Quant. Biol. 56, 177-186 and Meyerson, M., Enders, G. H., Wu, C.-L., Su, L.-K., Gorka, C., Nelson, C., Harlow, E. & Tsai, L.-H. (1992) EMBO J. 11, 2909-2917]. Two members of cdc2 family, p34cdc2 (also named cdk1) and cdk2, have been identified in mammalian cells. cdk1 kinase regulates the progression from G2 to M phase, and cdk2 kinase has been proposed to regulate the progression from G1 to S phase. In this work, we have cloned and structurally characterized a third member of the cdc2 kinase family with 58% amino acid sequence identity to mouse cdk1 and 61% identity to human cdk2. We call this kinase neuronal cdc2-like kinase (nclk) because, in contrast to either cdk1 or cdk2, nclk is expressed at high levels in terminally differentiated neurons no longer in the cell cycle. Previous studies have shown [Hisanaga, S., Kusubata, M., Okumura, E. & Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803 and Guan, R. J., Hall, F. L. & Cohlberg, J. A. (1992) J. Neurochem. 58, 1365-1371] that cdk1 kinase, but not other structurally defined protein kinases, could phosphorylate the repeated Lys-Ser-Pro (KSP) motifs found in mammalian high and middle molecular mass neurofilament subunits in vitro, but the precise molecular nature of the endogenous neuronal KSP kinase has remained undefined. The structural similarity of nclk to cdk1 kinase and its high level of expression in terminally differentiated neurons suggest that nclk may play a role in the phosphorylation of the neurofilament KSP repeats in vivo, a function distinct from cell cycle regulation.
Recombination in vitro between herpes simplex virus type 1 a sequences.
Authors: Bruckner R C , Dutch R E , Zemelman B V , Mocarski E S , Lehman I R
Published: May 2006

We have partially purified an activity from extracts of cells infected with herpes simplex virus type 1 that mediates recombination between repeated copies of the 317-base-pair a sequence of herpes simplex virus type 1. Recombination leads to deletion of a lacZ indicator gene situated between two directly repeated copies of the a sequence and is scored by transformation of lacZ- Escherichia coli. The two products of the reaction can be observed directly by restriction enzyme digestion and Southern blot analysis. The recombinase activity is also detectable, but at a lower level, in uninfected cell extracts. The DNA substrate must contain the two a sequences arranged in direct orientation to generate the lacZ deletion. However, when the a sequences are arranged in inverted orientation, an inversion results. A substrate with two homologous sequences of size and G + C content similar to the a sequence undergoes recombination at a much lower frequency. The reaction requires a divalent cation (Mg2+ or Mn2+) but not ATP or any other nucleoside triphosphate. The simple requirements and specificity for the a sequence suggest that the recombination may proceed by a site-specific mechanism.
Adoptive transfer of skin-selective autoimmunity induced by Skn alloantigenic disparities.
Authors: Jackman S H , Boyse E A , Goldberg E H
Published: May 2006

Two unlinked genes of the mouse, Skn-1 and Skn-2, each with alterative alleles, specify alternative cell-surface Skn alloantigens expressed only by epidermal and neural cells. C57BL/6 (B6) and A/J (A) strain mice differ at both Skn loci. Thus lethally irradiated B6 mice restored with (B6 x A)F1 hybrid hematopoietic cells [(B6 x A)/B6 chimeras] reject A strain (Skn-incompatible) skin grafts. Our studies were designed primarily to test the inference that (B6 x A)F1 lymphoid cells, after differentiating in B6 recipients, which lack the Skn alloantigens of A strain mice, may make an Skn-related, skin-selective autoimmune response when returned to their native (B6 x A)F1 habitat. Severe cutaneous lesions did, indeed, ensue after spleen cells of (B6 x A)/B6 chimeras were transferred to (B6 x A)F1 recipients, provided that three conditions were met--namely, (i) priming of the (B6 x A)/B6 chimeric donor by grafting and rejection of Skn-incompatible A strain skin grafts, (ii) stimulation of the recipient's skin as from shaving, at which sites the lesions were mainly located, and (iii) pretreatment of the (B6 x A)F1 recipients with cyclophosphamide or sublethal irradiation. Spleen cells of control female chimeras primed by grafting and rejection of H-Y (Skn-compatible) B6 male skin failed to incite the Skn-typical cutaneous lesions in (B6 x A)F1 recipients, indicating that these lesions were Skn-specific and not a nonspecific consequence of incompatible skin grafting per se. Normally compatible A strain skin grafts, but not Skn-compatible B6 skin grafts, were rejected by cyclophosphamide-treated (B6 x A)F1 recipients of (B6 x A)/B6 spleen cells from Skn-primed chimera donors. Treatment of primed chimeras' spleen cells with antiserum to H-2a (A strain) specifically abolished their capacity to adoptively incite the Skn-related autoimmune syndrome, confirming that the immune cells responsible are of (B6 x A)F1 origin and are not residual B6 derivatives. These findings add weight to the status of Skn systems as agents of tissue-selective histoincompatibility and, perhaps, of clinical disorders with a known or suspected autoimmune basis affecting the skin.
Regulation of an opioid-binding protein in NG108-15 cells parallels regulation of delta-opioid receptors.
Authors: Lane C M , Elde R , Loh H H , Lee N M
Published: May 2006

An opioid-binding protein has recently been purified from bovine brain and cloned, and its cDNA sequence has been obtained. Indirect evidence suggests that this protein has a role in opioid-receptor function. However, because direct testing of its function by expression of its cDNA has not yet been possible and because its structure bears no resemblance to G protein-coupled receptors, the role of this protein in opioid-receptor activity is still in question. An antibody raised to a portion of the predicted amino acid sequence of opioid-binding cell-adhesion molecule (OBCAM) specifically labeled the surface of NG108-15 cells, as visualized by immunofluorescence with confocal microscopy. Furthermore, chronic treatment of these cells with opioid agonist, which down-regulates opioid receptors, reduced OBCAM immunoreactivity (ir). Down-regulation of both opioid receptors and OBCAM-ir was greatest after chronic treatment of NG108-15 cells with delta-opioid agonists, as well as with nonselective agonists such as etorphine, whereas other agonists including [D-Ala2-N-MePhe4-Gly-ol]enkephalin, morphine, levorphanol, dynorphin A-(1-13), and U-50,488H were less effective or ineffective. Chronic treatment of NG108-15 cells with muscarinic agonists had no effect on OBCAM-ir. Furthermore, NG108-15 cells transfected with an antisense construct to OBCAM have a reduced density of opioid-binding sites as well as reduced OBCAM-ir. Taken together, these results strongly suggest that OBCAM has a role in opioid-receptor function in NG108-15 cells.
Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis.
Authors: Hayashi T , Fukuto J M , Ignarro L J , Chaudhuri G
Published: May 2006

Estradiol is known to exert a protective effect against the development of atherosclerosis, but the mechanism of this hormonal action is unknown. One of the early events in the development of atherosclerosis is the adhesion of macrophages to endothelial cells, and nitric oxide (NO) inhibits this process. We show that basal release of NO is greater with endothelium-intact aortic rings from female rabbits than those from males. Oophorectomy diminishes both circulating estradiol concentration and basal release of NO to levels seen in male rabbits. These data establish that basal NO release from endothelium-intact aortic rings depends on circulating estradiol concentration and offer an explanation for the protective effect of estradiol against the development of atherosclerosis.
Activin inhibits binding of transcription factor Pit-1 to the growth hormone promoter.
Authors: Struthers R S , Gaddy-Kurten D , Vale W W
Published: May 2006

Activin A is a potent growth and differentiation factor related to transforming growth factor beta. In somatotrophs, activin suppresses the biosynthesis and secretion of growth hormone (GH) and cellular proliferation. We report here that, in MtTW15 somatotrophic tumor cells, activin decreased GH mRNA levels and inhibited expression of transfected GH promoter--chloramphenicol acetyltransferase fusion genes. Deletion mapping of nucleotide sequences mediating this inhibition led to the identification of a region that has previously been characterized as binding the pituitary-specific transcription factor Pit-1/GHF-1. Characterization of nuclear factor binding to this region demonstrated that binding of Pit-1 to the GH promoter is lost on activin treatment. These results indicate that activin-induced repression of GH biosynthesis is mediated by the loss of tissue-specific transcription factor binding to the GH promoter and suggest a possible general mechanism for other activin responses, whereby activin regulates the function of other POU- or homeodomain-containing transcription factors.
Additional antitumor ecteinascidins from a Caribbean tunicate: crystal structures and activities in vivo.
Authors: Sakai R , Rinehart K L , Guan Y , Wang A H
Published: May 2006

Ecteinascidins (Ets), isolated from the Caribbean tunicate Ecteinascidia turbinata, protect mice in vivo against P388 lymphoma, B16 melanoma, M5076 ovarian sarcoma, Lewis lung carcinoma, and the LX-1 human lung and MX-1 human mammary carcinoma xenografts. Crystal structures of two tris(tetrahydroisoquinoline) Ets were investigated with single crystals of the 21-O-methyl-N12-formyl derivative of Et 729 and the natural N12-oxide of Et 743. Representatives of an additional class of Ets, Et 722 and Et 736, isolated from the same organism, were assigned tetrahydro-beta-carboline-substituted bis(tetrahydroisoquinoline) structures by NMR and fast atom bombardment MS spectra.
Modulation of synaptic gain by light.
Authors: Wu S M , Yang X L
Published: May 2006

Although synaptic transmission in the retina has been assumed to be static, it appears that the voltage gains of the synapses between photoreceptors and second-order cells can be enhanced by light. Voltage gains of the synapses between rods and bipolar (or horizontal) cells are about 10 times higher in the presence of dim background light than in darkness. This increase in synaptic gain may compensate for the loss of rod light responsiveness caused by weak background light so that the animal can maintain good rod sensitivity under moonlight or starlight, the natural lighting condition for mating and food catching.
Ectopic lymphokine gene expression in human peripheral blood lymphocytes in vivo.
Authors: Chambers C A , Kang J , Hozumi N
Published: May 2006

An animal model to study the effects of ectopic expression of cytokines involved in cell growth and differentiation has been established. Retrovirus vectors containing the human interleukin 6 cDNA were used to produce high titer virus-producing lines. Human peripheral blood lymphocytes (hPBLs) were successfully infected with the retrovirus and engrafted into severe combined immunodeficient mice. The majority of the animals were engrafted with hPBLs, as determined by the presence of human glucose phosphate isomerase. Furthermore, six of seven mice engrafted with hPBLs infected with high titer virus and detectable hPBLs present in the spleen expressed the retroviral human interleukin 6 gene. Importantly, human interleukin 6 protein was expressed at physiologically significant levels in these mice. These results demonstrate that models for human disease and immunotherapy involving retrovirus-mediated gene transfer into human cells can be developed in mice.
Conserved energy without work or heat.
Authors: Samuelson P A
Published: May 2006

By merely observing the common empirical temperature that two like subsystems in contact reach, as a recordable function of their initial empirical temperatures and specific volumes, we can deduce logically a conserved energy function that depends only on empirical temperature and specific volume. What makes possible this offbeat 18th-century inference, quite free of any 19th-century concepts of "heat" or measurable mechanical work, is the Invariance Law of nature, whereby any four like subsystems must settle down to the same common temperatures regardless of which two-by-two pairings are made-so to speak, an "independence of path" property. Mathematically, the Invariance Law imposes Abel-like functional equations on the admissible functions. Remarkably, recourse to such temperature-equalization data renders it impossible to identify one constant in the energy function. By observing how two quite different substances come into equilibrium, or by utilizing "adiabatic slopes" at two points for one substance, or by observing its expansion into a vacuum, we can determine all its unknowns. Much of conventional expositions of the First and Second Laws seems derivable from the equation of state and the First Law's energy function.
Industry as a metabolic activity.
Authors: Smart B
Published: May 2006

The concept of "industrial economic metabolism" can provide a bridge to better understanding between environmentalists and industry. In nature each individual or species reacts to natural stimuli, competing with others for resources, extending its domain until it loses comparative advantage and comes to equilibrium with an adjacent competitor. Those species that succeed over time flourish; those that do not, diminish or disappear. Nature's rule book has no moral or ethical ingredient beyond self-interest. Corporate metabolisms are remarkably similar to those of nature. They too react to stimuli, collect and use resources, and grow or perish based on how effectively they compete. Corporate management recognizes and responds naturally and efficiently to cost and price signals. Through them it selects resources and converts them into useful products. The efficiency with which this is done is measured by profit, the lifeblood of the corporation and its means of growth. Profit thus provides a discipline on corporate behavior, encouraging efficient performers, and, by its absence, weeding out others. Unfettered by influences other than economics, the path to corporate success is unlikely to be a compassionate one. The dilemma of the manager is that to do what is socially "right" often conflicts with what must be done to survive and prosper. Fortunately, corporations' behavior can be altered by society when their purely economic role comes into conflict with other human values. The environment and the economy are not separate systems but intertwined to form a complex natural and social setting. The human-designed economic system depends on natural resource inputs, and in turn its metabolic wastes can overload the ecological system, threatening the long-term survivability of both. Increasing concern for the environment now gives the farsighted manager new latitude. There are competitive benefits in some pollution prevention. But there are not sufficiently strong forces to correct all current ills. In addition, we must harness the metabolism of the industrial world to the realities of the natural one by recognizing the immense value of depletable natural resources and ecosystems. Considering these resources as "cheap" or "free" encourages their overuse. What is needed are adjustments that price these resources at their true longterm value. Corporations will respond naturally, quickly, and efficiently to such signals. If we can send our metabolic industry the economic signals it can understand, we can retrofit our human economic system to live in harmony with the natural ecosystem of which we are a part. If we do not, nature assuredly will not accommodate our failure by changing its ways.
Pollution prevention as a market-enhancing strategy: a storehouse of economical and environmental opportunities.
Authors: Hoffman J S
Published: May 2006

EPA's (Environmental Protection Agency) Green Lights Program for energy-efficient lighting illustrates the economic benefits and the market-transforming value of a pollution prevention philosophy. Using technologies available today, and assuming current prices, this program is expected to reduce air pollution 5%, while saving the nation's businesses up to 20 billion in electric bills every year. However, these pollution prevention and savings estimates may be low. As Green Lights transforms the market for lighting services by creating a higher demand for better technologies at lower costs, the program will likely achieve even larger pollution reductions and electricity savings.
Polymer recycling: opportunities and limitations.
Authors: Stein R S
Published: May 2006

The disposal of polymer solid waste by means other than landfilling is necessary. The various approaches-source reduction, incineration, degradation, composting, and recycling-all have their roles and must be employed in an integrated manner. Where appropriate, recycling has ecological advantages, but its application is dependent upon the feasibility of collection, sorting, and/or compatibilization of resulting mixtures to produce economically viable products. The practice should be encouraged by societal or legislative pressure which recognizes that the cost of disposal should be a factor in determining the cost of a product.
Industrial ecology: an emerging management science.
Authors: Piasecki B
Published: May 2006

Pollution is a complex equation, compounded by population, rate of consumption, and toxic emissions per unit of resource consumed. This article defines industrial ecology as a management science that focuses a corporation's expertise on the third variable. Thus, industrial ecology is involved with changing the efficiency of machines, not just changing the law or a firm's compliance strategy. This article also explores how this emphasis allows a profoundly different orientation than the end-of-the-pipe regulatory approach of the last 20 yr.
Friedel-Crafts phenylethylation of benzene and toluene with alpha- and beta-phenylethyl chlorides: pi-aryl participation in polarized donor-acceptor beta-phenylethylating complexes distinct from phenonium ions (sigma complexes).
Authors: Olah G A , Hamanaka S , Wilkinson J A , Olah J A
Published: May 2006

Friedel-Crafts alpha-phenylethylation of benzene and toluene, compared with beta-phenylethylation, shows a low ratio of ortho/para substitution with only 2-3% meta isomer formation, with kT/kB rate ratios between 58 and 76. The data indicate late arenium ion (sigma complex)-like transition states. beta-Phenylethylation in contrast gives low kT/kB rate ratios with a substantially higher ortho/para isomer ratio of 17-21% for the meta substituent. Experimental evidence points to thermodynamically controlled isomerizations effecting results. The data also indicate formation of an oriented pi-complex involving phenyl participation (as contrasted with complete phenonium ion formation) in the alkylation intermediates of the beta-phenylethylation reactions.
Structural analysis of a carcinogen-induced genomic rearrangement event.
Authors: Barr F G , Davis R J , Eichenfield L , Emanuel B S
Published: May 2006

We have explored the mechanism of genomic rearrangement in a hamster fibroblast cell culture system in which rearrangements are induced 5' to the endogenous thymidine kinase gene by chemical carcinogen treatment. The wild-type region around one rearrangement breakpoint was cloned and sequenced. With this sequence information, the carcinogen-induced rearrangement was cloned from the corresponding rearranged cell line by the inverse polymerase chain reaction. After the breakpoint fragment was sequenced, the wild-type rearrangement partner (RP15) was isolated by a second inverse polymerase chain reaction of unrearranged DNA. Comparison of the sequence of the rearrangement breakpoint with the wild-type RP15 and 5' thymidine kinase gene regions revealed short repeats directly at the breakpoint, as well as nearby A + T-rich regions in each rearrangement partner. Pulsed-field electrophoresis analysis demonstrated that this rearrangement is an interstitial deletion of 35 kilobases. Southern blot analysis of the RP15 region in unrearranged parental cells showed a demethylated CpG island and a complex of DNase I-hypersensitive sites adjacent to the breakpoint in the region deleted by the rearrangement. Therefore, these studies reveal interesting sequence and chromatin features near the rearrangement breakpoints and suggest a role for nuclear organization in the mechanism of carcinogen-induced genomic rearrangement.
Coexpression of mu and gamma 1 heavy chains can occur by a discontinuous transcription mechanism from the same unrearranged chromosome.
Authors: Nolan-Willard M , Berton M T , Tucker P
Published: May 2006

We previously documented that a single BCL1 leukemia cell can produce mu and gamma 1 immunoglobulin heavy chains with identical variable segments in an allelically excluded fashion without heavy chain constant region gene rearrangement. To understand the mechanism of dual mu/gamma 1 synthesis in BCL1 subclones, we have analyzed mature and pre-RNA at the nascent and steady-state levels. We find mu and gamma 1 sequences linked in pre-RNA. However, the primary mu and gamma 1 transcription units are about the same length (approximately 15 kilobases). Initiation of gamma 1 pre-RNA occurs upstream of C gamma 1 at sites identical to those seen in lipopolysaccharide/interleukin-4-induced normal B cells. We propose that dual mu/gamma 1 RNA synthesis occurs by a discontinuous transcription mechanism involving either trans-splicing or ligation of mu pre-RNA initiated 5' of the variable-diversity-joining region to gamma 1 pre-RNA initiated 5' of C gamma 1.
Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors.
Authors: Sun W , Ferrer-Montiel A V , Schinder A F , McPherson J P , Evans G A , Montal M
Published: May 2006

A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCR analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists with the following specificity sequence: domoate greater than kainate much greater than quisqualate greater than or equal to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid greater than or equal to L-glutamate much greater than N-methyl-D-aspartate. The kainate-elicited currents were specifically blocked by 6-cyano-7-nitroquinoxaline-2,3-dione but were insensitive to 2-amino-5-phosphonovalerate and kynurenic acid. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.
Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines.
Authors: Wicks I P , Wilkinson D , Salvaris E , Boyd A W
Published: May 2006

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The HEK protein was purified by using a IIIA4 antibody column and both N-terminal and internal amino acid sequences were obtained. A 51-mer degenerate oligonucleotide based on the internal amino acid sequence was used to screen an LK63-derived lambda gt10 cDNA library under low-stringency hybridization conditions. One clone of 2.5 kilobases (kb) was isolated and characterized and used to rescreen the library under more-stringent hybridization conditions. A 4.5-kb clone containing the entire HEK coding region was isolated and its complete DNA sequence was determined. The 4.5-kb insert was subcloned into the expression vector CDM8 and transfected into COS cells. COS cells transfected with the sense HEK/CDM8 construct stained specifically with the IIIA4 antibody, thereby confirming that the antigen recognized by the IIIA4 antibody and the expressed protein product of the HEK cDNA clone were identical. DNA sequence analysis revealed that HEK is a newly discovered member of the EPH/ELK family of receptor tyrosine kinases. Northern blot analysis of a number of cell lines demonstrated the expression of 5.5- to 6.0-kb HEK transcripts in LK63 and the T-cell lines JM and HSB-2. Southern blot analysis of DNA from LK63 suggested that the HEK gene was neither amplified nor rearranged in the LK63 tumor.
Characterization of an inversion on the long arm of chromosome 10 juxtaposing D10S170 and RET and creating the oncogenic sequence RET/PTC.
Authors: Pierotti M A , Santoro M , Jenkins R B , Sozzi G , Bongarzone I , Grieco M , Monzini N , Miozzo M , Herrmann M A , Fusco A
Published: May 2006

RET/PTC is a transforming sequence created by the fusion of the tyrosine kinase domain of the RET protooncogene with the 5' end of the locus D10S170 designated by probe H4 and is frequently found activated in human papillary thyroid carcinomas. RET and D10S170 have been mapped to contiguous regions of the long arm of chromosome 10: q11.2 and q21, respectively. To identify the mechanism leading to the generation of the oncogenic sequence RET/PTC, a combined cytogenetic and molecular analysis of several cases of papillary thyroid carcinomas was done. In four cases the results indicated that these tumors had RET/PTC activation and a paracentric inversion of the long arm of chromosome 10, inv(10)(q11.2q21), with breakpoints coincident with the regions where RET and D10S170 are located. Therefore, a chromosome 10q inversion provides the structural basis for the D10S170-RET fusion that forms the hybrid transforming sequence RET/PTC.
Peptide mimetics of the thrombin-bound structure of fibrinopeptide A.
Authors: Nakanishi H , Chrusciel R A , Shen R , Bertenshaw S , Johnson M E , Rydel T J , Tulinsky A , Kahn M
Published: May 2006

Recent work has suggested that the thrombin-bound conformation of fibrinopeptide A exhibits a strand-turn-strand motif, with a beta-turn centered at residues Glu-11 and Gly-12. Our molecular modeling analysis indicates that the published fibrinopeptide conformation cannot bind reasonably to thrombin but that reorientation of two residues by alignment with bovine pancreatic trypsin inhibitor provides a good fit within the deep thrombin cleft and satisfies all of the experimental nuclear Overhauser effect data. Based on this analysis, we have successfully designed and synthesized hybrid peptide mimetic substrates and inhibitors that mimic the proposed beta-turn structure. The results indicate that the turn conformation is an important aspect of thrombin specificity and that our turn mimetic design successfully mimics the thrombin-bound conformation of fibrinopeptide.
Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor.
Authors: Cull M G , Miller J F , Schatz P J
Published: May 2006

We have constructed a large library of random peptides fused to the C terminus of the lac repressor. The DNA binding activity of the repressor protein physically links the peptides to the plasmid encoding them by binding to lac operator sequences on the plasmid. This linkage allows efficient enrichment for specific peptide ligands in the random population of peptides by affinity purification of the peptide-repressor-plasmid complexes with an immobilized receptor. After transformation of Escherichia coli with recovered plasmids, the library can be amplified for additional rounds of affinity enrichment or specific plasmids can be sequenced to determine the primary structure of the peptides. We used a monoclonal antibody specific for the peptide dynorphin B as a model receptor to screen a random dodecamer library. After only two rounds of enrichment, the majority of the plasmids in the selected population encoded fusion peptides that bound specifically to the antibody. These peptides contain a consensus sequence similar to a segment of dynorphin B (RQFKVV). This technique should be useful to find peptide ligands for a variety of biological receptors.
Common double- and single-stranded DNA binding factor for a sterol regulatory element.
Authors: Stark H C , Weinberger O , Weinberger J
Published: May 2006

A cis-acting element necessary for sterol regulation, SRE-1, has previously been identified in the promoters of the low density lipoprotein receptor, hydroxymethylglutaryl (HMG)-CoA reductase, and HMG-CoA synthase genes. In this report we describe a nuclear factor, SRE-BF, isolated from Chinese hamster ovary nuclear extracts, that binds to the SRE-1 octanucleotide sequence. In addition to sequence-specific binding to SRE-1, as indicated by competition analysis with double-stranded DNA fragments, single-stranded oligomer DNA sequences also compete for binding in a sequence-specific fashion. Photochemical cross-linking experiments suggest that a common protein factor, with apparent molecular mass of 45-49 kDa, recognizes both single-stranded and double-stranded SRE-1. The binding specificity of SRE-BF to single-stranded SRE-1 closely correlates with the reported in vivo ability of SRE-1 to direct sterol responsiveness of transcription.
Nonrandom distribution of gp120 N-linked glycosylation sites important for infectivity of human immunodeficiency virus type 1.
Authors: Lee W R , Syu W J , Du B , Matsuda M , Tan S , Wolf A , Essex M , Lee T H
Published: May 2006

More than 20 consensus N-linked glycosylation sites occur in the gp120 coding sequence of most isolates of human immunodeficiency virus type 1. Based on the N-linked glycosylation pattern of a well-characterized recombinant gp120, it is likely that N-linked sugars are present at most, if not all, of the consensus glycosylation sites of the heavily glycosylated gp120. In this study, we evaluated the relative importance of each of the 24 N-linked glycosylation sites of gp120 in the molecular clone HXB2 to viral infectivity. The ability of HXB2-derived mutants, each having 1 of the 24 N-linked glycosylation sites mutated by site-directed mutagenesis, to infect CD4-positive SupT1 cells was compared with that of the wild-type virus. We found that most of the individual consensus N-linked glycosylation sites are dispensable for viral infectivity. The five consensus N-linked glycosylation sites that are likely to have important roles in infectivity are all located in the amino-terminal half of gp120, indicating that the N-linked glycosylation sites that are important for infectivity of human immunodeficiency virus type 1 are not randomly distributed in gp120. We predict that a partially glycosylated gp120 with most of the dispensable N-linked glycosylation sites removed may be a better vaccine candidate than the fully glycosylated gp120.
Identification of a specific inhibitor for DNA ligase I in human cells.
Authors: Yang S W , Becker F F , Chan J Y
Published: May 2006

A protein inhibitor for human DNA ligase I has recently been identified. It was copurified with a fraction of the enzymes from HeLa cells through several steps of chromatography. The inhibitor was first identified by the absence of ligation activity of the associated enzyme, while it retained the ability to form the ligase-[32P]AMP adducts. The inhibitor was eluted as a single peak at approximately 0.25-0.30 M NaCl from a Mono S column. It inhibited the ligation of both double-stranded and single-stranded breaks by purified DNA ligase I but not by T4 DNA ligase and DNA ligase II. Subsequent gel-filtration chromatography indicated that this inhibitor, with a molecular mass of 55-75 kDa, could form a complex with DNA ligase I and inhibited the DNA ligation activity. Rechromatography of the ligase I-inhibitor complex in high-salt conditions resulted in the dissociation of the complex and the restoration of enzyme activity, indicating that the physical interaction of inhibitor with DNA ligase I is one of the mechanisms of inhibition. These data indicate that this protein inhibitor for DNA ligase I may play a specific role in regulating DNA ligation during replication, repair, or recombination.
Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.
Authors: Davidson A L , Shuman H A , Nikaido H
Published: May 2006

Maltose transport across the cytoplasmic membrane of Escherichia coli is dependent on the presence of a periplasmic maltose-binding protein (MBP), the product of the malE gene. The products of the malF, malG, and malK genes form a membrane-associated complex that catalyzes the hydrolysis of ATP to provide energy for the transport event. Previously, mutants were isolated that had gained the ability to grow on maltose in the absence of MBP. After reconstitution of the transport complex into proteoliposomes, measurement of the ATPase activity of wild-type and mutant complexes in the presence and absence of MBP revealed that the wild-type complex hydrolyzed ATP rapidly only when MBP and maltose were both present. In contrast, the mutant complexes have gained the ability to hydrolyze ATP in the absence of maltose and MBP. The basal rate of hydrolysis by the different mutant complexes was directly proportional to the growth rate of that strain on maltose, a result indicating that the constitutive ATP hydrolysis and presumably the resultant cyclic conformational changes of the complex produce maltose transport in the absence of MBP. These results also suggest that ATP hydrolysis is not directly coupled to ligand transport even in wild-type cells and that one important function of MBP is to transmit a transmembrane signal, through the membrane-spanning MalF and MalG proteins, to the MalK protein on the other side of the membrane, so that ATP hydrolysis can occur.
Chlorambucil-induced mutations in mice recovered in homozygotes.
Authors: Flaherty L , Messer A , Russell L B , Rinchik E M
Published: May 2006

Chlorambucil induced a number of recessive visible mutations in the mouse. Induction of these mutations was studied in two mating schemes, each designed to recover mutations after two intercrosses. In scheme 1, 10 mutations were detected in 82 mice; in scheme 2, 1 mutation was detected in 19 mice. We have estimated that the proportion of gametes carrying a recessive visible mutation may be as high as 25% after a dose of 10 mg of chlorambucil per kg to early spermatids. Seven of these mutations caused morphologically distinct abnormalities, including (i) a cerebellar abnormality similar to that expressed in homozygotes for the reeler (rl) mutation; (ii) open eyelids at birth; (iii) a rostral head hemangioma; (iv) abnormally small spleens, anemia, and umbilical hemorrhages; (v) immobility at birth; (vi) polycystic kidneys; and (vii) a circling behavior. Four additional mutations resulted in growth retardation and a runting syndrome. Because, in earlier studies, all molecularly characterized mutations induced by chlorambucil in poststem cells have proved to be deletions, these recessive visible mutations are probably deletions as well. These mutations may be useful in isolating and characterizing the genes responsible for the observed phenotypes.
Functional analysis of a type IIB von Willebrand disease missense mutation: increased binding of large von Willebrand factor multimers to platelets.
Authors: Cooney K A , Lyons S E , Ginsburg D
Published: May 2006

Type IIB von Willebrand disease is an autosomal dominant bleeding disorder characterized by the selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma, presumably due to their abnormally increased reactivity with platelets. We and others have recently identified a panel of missense mutations clustered in the platelet glycoprotein Ib binding domain of vWF from patients with type IIB von Willebrand disease. We now report functional analysis of one of the most frequent type IIB missense mutations, Arg-543----Trp (vWF R543W). vWF from a human umbilical vein endothelial cell culture heterozygous for the vWF R543W mutation showed markedly increased binding of large vWF multimers to platelets in the presence of a low dose of ristocetin compared to vWF from a normal control culture. Recombinant vWF containing the vWF R543W mutation expressed in COS-7 cells also demonstrated increased binding of large vWF multimers. Mixed multimers obtained by cotransfection of mutant and wild-type cDNAs showed partial dominance of the vWF R543W mutation. Thus these data demonstrate that the vWF R543W mutation alone is sufficient to confer increased binding of large vWF multimers to platelets in a dominant fashion and that no other factors relating to vWF posttranslational processing or secretion in endothelial cells are required for this effect.
Hox-1.11 and Hox-4.9 homeobox genes.
Authors: Nazarali A , Kim Y , Nirenberg M
Published: May 2006

Mouse Hox-1.11 and Hox-4.9 genes were cloned, and the nucleotide sequences of the homeobox regions were determined. In addition, nucleotide sequence analysis of the homeobox regions of cloned Hox-4.3 and Hox-4.2 genomic DNA revealed some differences in nucleotide sequences and in the deduced homeodomain amino acid sequences compared with the sequences that have been reported.
Expression of alternatively spliced human T-lymphotropic virus type I pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers.
Authors: Berneman Z N , Gartenhaus R B , Reitz M S , Blattner W A , Manns A , Hanchard B , Ikehara O , Gallo R C , Klotman M E
Published: May 2006

Although human T-cell lymphotropic virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), the role of viral gene expression in the progression to and maintenance of the leukemic state in vivo is unclear because of the inability of most previous studies to readily detect HTLV-I RNA in infected individuals. By using the reverse transcriptase-polymerase chain reaction, we detected spliced messages for the HTLV-I pX regulatory genes in primary uncultured cells from ATL patients and healthy asymptomatic carriers. In addition to the expected doubly spliced pX message, three alternatively spliced mRNAs were demonstrated (pX delta 17, pX-p21rex, and pX-orfII mRNAs, where orf = open reading frame). The same splice sites were shown in the messages from uncultured ATL cells and from the HTLV-I-producing C10/MJ cell line. Alternatively spliced pX mRNAs have the potential to code for known and putative pX gene products. Among the transcripts is a monocistronic mRNA likely to code for p21rex (pX-p21rex mRNA). Since alternative splicing of HTLV-I pX mRNA can be found in primary uncultured cells, it is likely to have a functional significance in vivo. This suggests possible roles for HTLV-I gene expression in the progression to and maintenance of ATL, as well as in the phase preceding it.
Purification, molecular cloning, and functional expression of the cholecystokinin receptor from rat pancreas.
Authors: Wank S A , Harkins R , Jensen R T , Shapira H , de Weerth A , Slattery T
Published: May 2006

The cholecystokinin (CCK) family of peptides and their receptors are widely distributed throughout the gastrointestinal and central nervous systems where they regulate secretion, motility, growth, anxiety, and satiety. The CCK receptors can be subdivided into at least two subtypes, CCKA and CCKB on the basis of pharmacological studies. We report here the purification of the CCKA receptor to homogeneity from rat pancreas by using ion-exchange and multiple affinity chromatographic separations. This allowed partial peptide sequencing after chemical/enzymatic cleavage and synthesis of degenerate oligonucleotide primers. These primers were used for initial cloning of the cDNA from rat pancreas by PCR. The predicted protein sequence of the cDNA clone contained the five partial peptide sequences obtained from the purified protein. Seven putative transmembrane domains suggest its membership in the guanine nucleotide-binding regulatory protein-coupled receptor superfamily. In vitro transcripts of the cDNA clone were functionally expressed in Xenopus oocytes and displayed the expected agonist and antagonist specificity.
Human combinatorial antibody libraries to hepatitis B surface antigen.
Authors: Zebedee S L , Barbas C F , Hom Y L , Caothien R H , Graff R , DeGraw J , Pyati J , LaPolla R , Burton D R , Lerner R A
Published: May 2006

Human antibody Fab fragments that bind to hepatitis B surface antigen (HBsAg) were generated by using a recombinant phage surface-display expression system. Characterization of HBsAg-specific Fab fragments isolated from two vaccinated individuals reveals diversity in specificity of antigen binding and in the sequences of the complementarity-determining region. The sequence results show examples of human light-chain promiscuity that result in fine specificity changes and a strong relationship to a human germ-line gene. This application illustrates further that this technique is a powerful tool to isolate distinct human antibodies against immunogenic viral targets.