bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of the chicken progesterone receptor.
Authors: Jeltsch J M , Krozowski Z , Quirin-Stricker C , Gronemeyer H , Simpson R J , Garnier J M , Krust A , Jacob F , Chambon P
Published: May 2006

Monospecific antibodies directed against the chicken progesterone receptor (PR) form B were used to screen a randomly primed phage lambda gt11 cDNA expression library prepared from size-fractionated chicken oviduct mRNA. Two independent immunoreactive clones, lambda cPR1 and lambda cPR2, were isolated. Antibodies selected from anti-PR form B antiserum on matrices of lambda cPR1 and lambda cPR2 fusion proteins detected two proteins on electrophoretic immunoblots of crude and purified PR preparations. These proteins had the same apparent molecular weights as did PR forms A and B crosslinked with the tritiated progestin R 5020. Thus, lambda cPR1 and lambda cPR2 fusion proteins contain epitopes present in both PR forms A and B. A cDNA clone, lambda cPR3, containing the inserts of both lambda cPR1 and lambda cPR2, was isolated from a randomly primed lambda gt10 oviduct cDNA library, indicating that both cDNA inserts were derived from the same oviduct mRNA. Additional evidence that these cDNAs correspond to PR mRNA was provided by sequencing the lambda cPR3 cDNA insert, since it was found to encode the sequence of three tryptic peptides prepared from purified PR form B. A fourth and a fifth cDNA clone, lambda cPR4 and lambda cPR5, were sequentially isolated from the same lambda gt10 cDNA library beginning with a probe derived from the 3' end of the lambda cPR3 insert. Partial DNA sequencing of lambda cPR4 and lambda cPR5 revealed the presence of a sequence coding for a cysteine-rich domain that is strikingly homologous to the amino acid sequences present in the putative DNA-binding domain of the human and chicken estrogen receptors, human glucocorticoid receptor, and v-erbA gene product of the avian erythroblastosis virus.
recD: the gene for an essential third subunit of exonuclease V.
Authors: Amundsen S K , Taylor A F , Chaudhury A M , Smith G R
Published: May 2006

Exonuclease V (EC 3.1.11.5) of Escherichia coli, an enzyme with multiple activities promoting genetic recombination, has previously been shown to contain two polypeptides, the products of the recB and recC genes. We report here that the enzyme contains in addition a third polypeptide (alpha) with a molecular mass of about 58 kDa. The alpha polypeptide is not synthesized by a class of mutants (previously designated recB) lacking the nuclease activity of exonuclease V but retaining recombination proficiency. The gene, recD, coding for the alpha polypeptide is located near recB in the order thyA-recC-ptr-recB-recD-argA on the E. coli chromosome. The recB and recD genes appear to be governed by a common promoter to the left of recB; a weaker promoter appears to govern recD alone. In the light of these results we discuss the relation between the structure and function of the three polypeptides of exonuclease V, hereby alternatively designated RecBCD enzyme.
Localization and sequence of a vaccinia virus gene required for multiplication in human cells.
Authors: Gillard S , Spehner D , Drillien R , Kirn A
Published: May 2006

A vaccinia virus host range mutant (hr mutant) deleted of 18 kilobase pairs at the left end of the genome has been employed to precisely map a viral gene required for multiplication in human cells. DNA fragments from the wild-type virus were inserted into the thymidine kinase gene of mutant virus by means of in vivo homologous recombination, and the recombinants obtained were screened for their ability to multiply in human cells. A short sequence, 855 base pairs long, overlapping the HindIII M and K fragments was able to restore a normal host range on the mutant virus. A single long open reading frame that could encode a polypeptide of 32.5 kDa was found in the nucleotide sequence of the host range gene. The direction of transcription and the length of the open reading frame are in excellent agreement with previous mapping of mRNAs within this region of the genome. In vitro translation of infected cell early mRNA, selected by hybridization to the host range gene, yielded a prominent polypeptide product whose size (29 kDa) was close to that expected from the predicted amino acid sequence. The phenotype of the hr mutant suggests that the host range gene plays a role in maintaining a high level of protein synthesis in human cells. It may behave positively by complementing the lack of an analogous cellular activity or negatively by antagonizing a cell function that inhibits viral multiplication.
Photoaffinity labeling demonstrates binding between Ia molecules and nominal antigen on antigen-presenting cells.
Authors: Phillips M L , Yip C C , Shevach E M , Delovitch T L
Published: May 2006

We have used radioiodinated photoreactive bovine insulin as antigen to examine the molecular nature of immunogenic complexes that form on antigen-presenting cells. The probe was allowed to bind to either insulin-presenting B-hybridoma cells, lipopolysaccharide-stimulated blasts, or bovine insulin-specific helper-T-hybridoma cells in the dark. Samples were then exposed to light to induce crosslinkage, solubilized, and analyzed by gel electrophoresis. Two protein bands at about 36 kDa and 27 kDa were specifically labeled on antigen-presenting cells but not on helper T cells. Treatment of these bands with dithiothreitol or endo-beta-N-acetylglucosaminidase F showed that each is composed of a single glycoprotein. These proteins are immunoprecipitable with haplotype-specific but not control anti-Ia antibodies. This identifies the labeled bands as the alpha and beta subunits of class II major histocompatibility antigens. We conclude that a molecular complex may form between Ia and antigen on antigen-presenting cells and that formation of this complex does not require the presence of a helper-T-cell antigen receptor.
Cholera toxin inhibits the T-cell antigen receptor-mediated increases in inositol trisphosphate and cytoplasmic free calcium.
Authors: Imboden J B , Shoback D M , Pattison G , Stobo J D
Published: May 2006

The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.
Calcitonin gene-related peptide: functional role in cerebrovascular regulation.
Authors: McCulloch J , Uddman R , Kingman T A , Edvinsson L
Published: May 2006

Distribution studies disclosed that all major cerebral arteries and cortical arterioles of the cat were invested with fine varicose nerve fibers that contained calcitonin gene-related peptide (CGRP)-like immunoreactivity; the trigeminal ganglia likewise contained CGRP immunoreactivity. Sequential immunostaining with antibodies to CGRP and to substance P (SP) revealed identical distributions of these two peptides in trigeminal ganglia and cerebrovascular nerve fibers, suggesting that CGRP and SP are colocalized in these nerves. CGRP completely disappeared from ipsilateral blood vessels after unilateral section of the trigeminal nerve. Exogenous CGRP was a potent relaxant of feline middle cerebral arteries in vitro (maximum relaxation, 10.5 +/- 1.5 mN; concentration eliciting half-maximal response, 9.6 +/- 1.3 nM). Perivascular microapplication of CGRP to individual cortical arterioles of chloralose-anesthetized cats provoked dose-dependent dilatations (maximum increase in diameter, 38 +/- 5%; concentration eliciting half-maximal response, approximately equal to 3 nM). CGRP was significantly more potent than SP as a cerebrovascular dilator, both in vitro and in situ. Chronic division of the ipsilateral trigeminal nerve in cats did not modify the magnitude of arteriolar responses to perivascular microapplication of either vasoconstrictor or vasodilator agents, but the duration of vasoconstrictor responses to norepinephrine (0.1 mM) or alkaline solutions (pH 7.6) was significantly increased. The cerebrovascular trigeminal neuronal system, in which CGRP is the most potent vasoactive constituent, may participate in a reflex or local response to excessive cerebral vasoconstriction that restores normal vascular diameter.
Membrane lipid phase as catalyst for peptide-receptor interactions.
Authors: Sargent D F , Schwyzer R
Published: May 2006

Catalysis of ligand-receptor interactions is proposed as an important function of the lipid phase of the cell membrane. The catalytic mechanism is deduced from observed specific interactions of amphiphilic peptides with artificial lipid bilayers. In our model a direct ligand-receptor reaction is replaced by multiple sequential steps including surface accumulation of charged ligands, ligand-membrane interactions, and ultimately binding to the receptor itself. By dividing the total free energy of binding among several steps, the energy per step, including the intrinsic receptor interaction energy, is kept to moderate values. The model thereby yields simple explanations for the large apparent association constants, the high association and dissociation rates, and the heterogeneity of binding sites so frequently found with pharmacological and biochemical ligand-receptor interactions. Furthermore, the measured apparent association constant is a function of the whole system rather than just the receptor. The same, fully functional receptor may show different binding characteristics in different surroundings, such as in another tissue or in a reconstituted system.
Rat cellular retinol-binding protein II: use of a cloned cDNA to define its primary structure, tissue-specific expression, and developmental regulation.
Authors: Li E , Demmer L A , Sweetser D A , Ong D E , Gordon J I
Published: May 2006

The primary structure of rat cellular retinol-binding protein (CRBP) II has been determined from a cloned cDNA. Alignment of this 134-amino acid, 15,580-Da polypeptide with rat CRBP revealed that 75 of 133 comparable residues are identical. Both proteins contain four tryptophan residues, which occupy identical relative positions in the two primary structures, providing a structural explanation for their similar fluorescence spectra when complexed to retinol. Two of the three cysteines in each single-chain protein are comparably positioned. Both polypeptides contain reactive thiol groups, but the rate of disruption of CRBP II-retinol complexes by p-chloromercuribenzoate is greater than that of CRBP-retinol. The small intestine contains the highest concentrations of CRBP II mRNA in adult rats. CRBP II mRNA is first detectable in intestinal RNA during the 19th day of gestation, a time that corresponds to the appearance of an absorptive columnar epithelium. Unlike in intestine, a dramatic fall in liver CRBP II mRNA concentration occurs immediately after birth. The CRBP II gene remains quiescent in the liver during subsequent postnatal development. These data suggest that ligand-protein interactions may be somewhat different for the two rat CRBPs. They also support the concept that CRBP II plays a role in the intestinal absorption or esterification of retinol and suggest that changes in hepatic metabolism of vitamin A occur during development.
Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein.
Authors: Birnbaum M J , Haspel H C , Rosen O M
Published: May 2006

Antibody raised against the human erythrocyte glucose transporter identified a recombinant lambda gt11 bacteriophage in a cDNA library prepared from immunoselected polysomal RNA from adult rat brain. The cDNA predicts a 492-amino acid protein that demonstrates 97.6% identity to the human hepatoma hexose carrier. The tissue distribution of the transporter mRNA is identical to that of immunologically identifiable protein and transport activity, except in liver in which high levels of transport are associated with little or no transporter mRNA or protein. As assayed by blot-hybridization analysis, mRNA from insulin-responsive and nonresponsive tissues are indistinguishable. These data suggest that a genetically unrelated protein is responsible for hexose transport in normal liver.
Identification of phosphoproteins correlated with proliferation and cell cycle arrest in Saccharomyces cerevisiae: positive and negative regulation by cAMP-dependent protein kinase.
Authors: Tripp M L , Piñon R , Meisenhelder J , Hunter T
Published: May 2006

Recent genetic and biochemical studies of two mutants of the cAMP pathway in yeast, cyr1 and bcy1, have demonstrated that cAMP-dependent protein phosphorylation plays a major regulatory role in the control of proliferation and differentiation. As a first step in examining this regulatory system in more detail and in identifying the protein substrates of cAMP-dependent protein kinase, we have analyzed phosphoprotein patterns in the mutants cyr1-2(ts) and bcy1 by two-dimensional polyacrylamide gel electrophoresis. Our analysis has revealed several proteins whose phosphorylation is controlled positively or negatively by the cAMP pathway in yeast. The presence of some of these phosphoproteins was directly associated with proliferation (positive regulation), while that of others was correlated with cell cycle arrest (negative regulation). The phosphoprotein patterns of cyr1-2(ts) temperature-arrested cells, and nitrogen (NH+4)-starved cells, were strikingly similar, suggesting that response to NH+4 is mediated in part by adenylate cyclase. Phosphoproteins whose presence correlated with cell cycle arrest were found to be phosphorylated on serine and threonine residues, while the major phosphoproteins present predominantly in proliferating cells were phosphorylated only on serine residues. None of the greater than 20 phosphoproteins we examined contained phosphotyrosine under either growth condition.
Meiotic disjunction of circular minichromosomes in yeast does not require DNA homology.
Authors: Mann C , Davis R W
Published: May 2006

Circular plasmids containing an autonomously replicating sequence (ARS) and a centromeric DNA sequence (CEN) segregate as independent linkage groups during meiosis in Saccharomyces cerevisiae. If two genetically marked plasmids are present in the same diploid cell, their segregation during meiosis may be determined relative to each other. It has been observed that for centromere plasmids containing some DNA sequences in common, these plasmids tend to segregate away from each other at the first meiotic division [Clarke, L. & Carbon, J. (1980) Nature (London) 287, 504-509; Clarke, L., Fitzgerald-Hayes, M., Buhler, J.-M. & Carbon, J. (1981) Stadler Genet. Symp. 13, 9-23]. Here we show that nonhomologous plasmids, having no detectable DNA sequence cross-hybridization, also tend to disjoin from each other at the first meiotic division. Therefore, this nonrandom segregation to opposite poles can occur by mechanisms that do not involve DNA sequence homology. This process may be an active nonhomologous pairing system or it may reflect unknown physical restraints on the meiotic segregation of the two plasmids. In either case, this process cannot be used as a possible assay for homologous meiotic pairing.
Genes for the eight ribosomal proteins are clustered on the chloroplast genome of tobacco (Nicotiana tabacum): similarity to the S10 and spc operons of Escherichia coli.
Authors: Tanaka M , Wakasugi T , Sugita M , Shinozaki K , Sugiura M
Published: May 2006

The nucleotide sequence of a tobacco (Nicotiana tabacum) chloroplast gene cluster that encodes eight proteins homologous to Escherichia coli ribosomal proteins L23, L2, S19, L22, S3, L16, L14, and S8 has been determined. RNA gel blot hybridization revealed that all eight coding regions are expressed in the chloroplasts. The arrangement of the eight genes resembles that found in the E. coli S10 and spc operons. Among the eight genes, the L2 and L16 genes contain 666- and 1020-base-pair introns, respectively. These intron boundary sequences are consistent with the conserved boundary sequences of the chloroplast group III introns [Shinozaki, K., Deno, H., Sugita, M., Kuramitsu, S. & Sugiura, M. (1986) Mol. Gen. Genet. 202, 1-5].
Chemical and functional correlates of postischemic renal ATP levels.
Authors: Stromski M E , Cooper K , Thulin G , Gaudio K M , Siegel N J , Shulman R G
Published: May 2006

Renal energy metabolism was investigated before, during, and after ischemic insults of varying durations with in vivo 31P NMR spectroscopy. The postischemic recovery of renal ATP was found to be a biphasic process regardless of the length of the ischemia. This two-stage recovery consisted of a quick initial component immediately upon reflow followed by a slower, more gradual return toward preischemic levels. To characterize the source of each phase of the recovery, kidneys were extracted with perchloric acid at the end of the different periods of ischemia (before reflow). Concentrations of adenine nucleotides and breakdown products adenosine, inosine, and hypoxanthine were determined by 1H NMR spectroscopy. Excellent correlation was found between the residual nucleotide pool and the magnitude of the initial phase of ATP recovery. Additionally, the renal ATP content after 120 min of reflow was shown to have a strong correlation with subsequent functional recovery. These experiments show that in vivo 31P NMR can provide new and dynamic information concerning the biochemical recovery from ischemia. Furthermore, this data has the potential to predict the eventual functional recovery of the organ.
In situ hybridization to localize mRNA encoding the neurotransmitter synthetic enzyme glutamate decarboxylase in mouse cerebellum.
Authors: Wuenschell C W , Fisher R S , Kaufman D L , Tobin A J
Published: May 2006

Glutamate decarboxylase (GAD; EC 4.1.1.15) is responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). We have used a cDNA sequence encoding GAD to produce a single-stranded RNA hybridization probe for GAD mRNA. This probe detects GAD mRNA in individual cells in sections of mouse cerebellum. The specificity of in situ hybridization with this probe rests on four criteria: the distribution of labeled cells matched the results we and others obtain with GAD immunohistochemistry (Purkinje, Golgi II, stellate, and basket neurons were labeled, whereas granule cells and glia were not); a negative control probe having a sequence identical to GAD mRNA did not specifically label any cerebellar cells; prior treatment of the sections with RNase abolished specific labeling; the labeling showed the melting behavior typical of nucleic acid hybrids. Translation of GAD mRNA is apparently restricted to neuronal cell bodies since GAD mRNA was detectable in neuronal perikarya but not in terminals. Also, the choice of GABA as a neurotransmitter appears to be made at the level of transcription since granule neurons did not contain detectable GAD mRNA. The level of GAD mRNA varied among the classes of neurons as well as from cell to cell within each neuron type.
Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end.
Authors: Beaton A R , Krug R M
Published: May 2006

The first step in the replication of influenza virion RNAs is the synthesis of full-length transcripts of these RNAs. The synthesis of these transcripts, or template RNAs, requires: unprimed initiation rather than the capped RNA-primed initiation used during viral mRNA synthesis, and antitermination at the polyadenylylation site used during mRNA synthesis. To determine the mechanism of template RNA synthesis, we prepared nuclear extracts from infected cells that were active in the synthesis of both template RNAs and viral mRNAs. By providing the dinucleotide ApG as primer, we circumvented the inefficient unprimed initiation catalyzed by these extracts and, as a consequence, were able to focus on the antitermination step. Antitermination, and hence template RNA synthesis, occurred when ApG but not a capped RNA was used as primer, indicating that the presence of a 5' capped end blocked antitermination at the 3' end of the transcript. Ultracentrifugation of the nuclear extract yielded a pellet fraction that contained viral nucleocapsids active in viral mRNA synthesis but not template RNA synthesis and a supernatant fraction that contained the antitermination factor. When the supernatant, which had essentially no activity by itself, was added to the pellet in the presence of ApG, template RNA synthesis was restored. Depletion experiments in which this supernatant was incubated with protein A-Sepharose containing antibodies to individual viral proteins demonstrated that the viral nucleocapsid protein was required for antitermination. The implications of these results for the control of viral RNA replication are discussed.
Reduction of the toxicity and mutagenicity of alkylating agents in mammalian cells harboring the Escherichia coli alkyltransferase gene.
Authors: Brennand J , Margison G P
Published: May 2006

The toxic, mutagenic, and carcinogenic effects of alkylating agents have been attributed to their ability to damage DNA. Reaction at the O6 position of guanine results in miscoding during DNA replication, has been shown to be mutagenic in both bacteriophage and bacteria, and may be responsible for malignant transformation. In common with many other prokaryotes and eukaryotes the Escherichia coli B strain contains a protein that repairs O6-alkylation damage in DNA by transferring the alkyl group to one of its own cysteine residues. We have recently cloned the E. coli O6-alkylguanine alkyltransferase gene and shown it to encode a 37-kDa protein containing an additional activity that removes alkyl groups from alkylphosphotriesters in DNA. To examine the biological effects of this gene in mammalian cells, we have now inserted the coding sequence into a retrovirus-based selectable expression vector and transfected it into Chinese hamster V79 cells that lack endogenous alkyltransferase activity. A clone expressing high levels of the bacterial protein was selected and shown to produce a 37-kDa alkyltransferase protein and to rapidly repair O6-methylguanine produced in the host genome following exposure to N-methyl-N-nitrosourea. In comparison with a control population, this clone is considerably more resistant to the toxic and mutagenic effects of alkylating agents that react extensively with oxygen atoms in DNA. The usefulness of these clones in examining the role of DNA alkylation and other biological effects of alkylating agents is discussed.
Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells.
Authors: Saraste J , Palade G E , Farquhar M G
Published: May 2006

The effect of temperature on secretory protein transport was studied by cell fractionation of rat pancreatic lobules, pulse-labeled in vitro with [35S]methionine and chased for 60 min at 16, 20, or 37 degrees C. Chase at 37 degrees C allowed secretory proteins to reach a zymogen granule fraction, whereas chase at 16 or 20 degrees C led to their extensive retention in a total microsomal fraction. To pinpoint the sites of transport inhibition, total microsomes were subfractionated by flotation in a sucrose density gradient. Five bands were resolved, of which the heaviest or B1 (density = 1.20 g/ml) consisted primarily of rough microsomes. The lighter fractions, B2 (1.17 g/ml), B3 (1.15 g/ml), and B4 (1.14-1.13 g/ml), consisted primarily of smooth vesicles derived from Golgi elements. B4 had the highest specific activity for galactosyltransferase, a trans Golgi cisternal marker; B2, B3, and B4 are assumed to represent cis, middle, and trans Golgi subcompartments, respectively. At the end of a 2-min pulse, a single peak of labeled proteins colocalized with B1. During subsequent 60-min chases, labeled proteins advanced to B2 at 16 degrees C and to B3 at 20 degrees C. At 37 degrees C the radioactivity remaining in the total microsomal fraction was distributed among four peaks (B1-B4). The results indicate that transport from the endoplasmic reticulum to the Golgi complex is strongly inhibited below 20 degrees C. At 16 degrees C, the bulk of the cohort of labeled secretory proteins is still in the rough endoplasmic reticulum, but its advancing front reaches cis Golgi elements. At 20 degrees C, the front advances to a middle Golgi compartment, and at 37 degrees C most of the cohort (approximately 70%) reaches condensing vacuoles and zymogen granules. It is concluded that transport steps along the endoplasmic reticulum-plasmalemma pathway have distinct temperature requirements.
Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures.
Authors: Nakamura T , Teramoto H , Ichihara A
Published: May 2006

A growth factor (HGF) stimulating DNA synthesis of adult rat hepatocytes in primary culture was found in rat platelets. HGF was purified from rat platelets to homogeneity by a three-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography on a Mono S column, and heparin-Sepharose chromatography. HGF was clearly distinguishable from the platelet-derived growth factor (PDGF) by fast protein liquid chromatography. HGF was a heat- and acid-labile cationic protein that was inactivated by reduction with dithiothreitol. Its molecular mass was estimated to be 27 kDa by NaDodSO4/PAGE and its amino acid composition was very different from that of PDGF. The purified HGF stimulated DNA synthesis in adult rat hepatocytes at 2 ng/ml and was maximally effective at 20 ng/ml; its effect was additive or synergistic with those of insulin and EGF, depending on their combinations. HGF did not stimulate DNA synthesis of Swiss 3T3 cells, while PDGF did not stimulate that of hepatocytes. Thus, HGF showed clearly different cell specificity from PDGF in its growth-promoting activities. These findings indicate that HGF is a growth factor in platelets for mature hepatocytes.
Fibrinogen mitogenic effect on hemopoietic cell lines: control via receptor modulation.
Authors: Levesque J P , Hatzfeld A , Hatzfeld J
Published: May 2006

The possibility that the mitogenic effect of fibrinogen, a major plasma protein (3 mg/ml), is mediated by specific membrane receptors was studied. Specific binding analysis showed that fibrinogen receptors are present only on hemopoietic cell lines that respond to its mitogenic effect. The mitogenic fibrinogen receptor is not recognized by antibodies specific for the platelet fibrinogen receptor or is not competitively blocked by synthetic peptides containing the Arg-Gly-Asp sequence, which is common to fibronectin, fibrinogen, vitronectin, and other cell-attachment proteins. The lymphoma-derived pre-B-cells (Raji) have 149,000 receptors, whereas the lymphoma-derived T cells (JM), which are 3 times smaller, have 54,000 receptors. These receptors have a Kd of 2 X 10(-7) M. They are inducible by stimuli specific for the cell lineage: activators of the breakdown of phosphatidylinositol phosphates, such as platelet activating factor for Raji cells, and adenylate cyclase agonists and cAMP analogues for JM cells. The stimuli have no mitogenic effect in the absence of fibrinogen; they do not change the Kd. Each stimulus increases the number of fibrinogen receptors in a dose-dependent manner, which correlates strongly (r = -0.98, n = 5) with an increased growth rate of cells in the presence of fibrinogen. This correlation concludes that the mitogenic effect of fibrinogen is controlled via receptor modulation.
An Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA5) partly encoded by the transformation-associated Bam WYH region of EBV DNA: preferential expression in lymphoblastoid cell lines.
Authors: Dillner J , Kallin B , Alexander H , Ernberg I , Uno M , Ono Y , Klein G , Lerner R A
Published: May 2006

Four peptides were synthesized on the basis of amino acid sequences deduced from a highly spliced transcript encoded by the Bam W, Y, and H fragments of the Epstein-Barr virus (EBV) genome [Bodescot, M., Chambraud, J. B., Farrell, P. J. & Perricaudet, M. (1984) EMBO J. 3, 1913-1917]. Rabbit antisera against three of the four peptides identified a nuclear polypeptide that varied between 22 and 70 kDa in molecular size. Four of 20 EBV-positive human sera contained antibodies against this polypeptide. Since this is the fifth EBV-determined nuclear antigen (EBNA) discovered in growth-transformed cells, it is designated EBNA5. The antigen was detected in virus nonproducer lines (less than 0.01% EBV early antigen expression) and is thus not dependent on the viral cycle. It was differentially expressed depending on the origin of the lines. All 10 lymphoblastoid cell lines tested expressed EBNA5, but it could not be detected in 10 of 11 EBV-carrying Burkitt lymphoma lines. Infection of tonsillar lymphocytes with the B95-8 strain of EBV induced six EBNA5-specific polypeptides that varied between 41 and 70 kDa in molecular size with regular increments of 6 kDa. This may be due to the fact that the EBNA5 coding sequence includes the Bam W internal repeat. Parallel infection of the EBV-negative Burkitt lymphoma line Ramos with the same viral substrain did not induce detectable levels of EBNA5, nor was this antigen present in permanently EBV-converted Ramos sublines. These findings imply that the expression of the viral genome varies among B cells having different phenotypes.
Purification, biochemical characterization, and biological function of human esterase D.
Authors: Lee W H , Wheatley W , Benedict W F , Huang C M , Lee E Y
Published: May 2006

Human esterase D (carboxylesterase; carboxylic-ester hydrolase, EC 3.1.1.1), a genetic marker of retinoblastoma, was purified to biochemical homogeneity from erythrocytes. The purification scheme including carboxymethylcellulose, phenyl-Sepharose, chromatofocusing, and hydroxylapatite chromatographies resulted in a 10,000-fold purification of the enzyme with 15% recovery of total activity. The Km of esterase D was estimated to be 10 X 10(-6) M using 4-methylumbelliferyl acetate as substrate. The enzymatic activity was inhibited by p-chloromercuribenzoate and HgCl2, suggesting an important role of SH group(s) in enzyme function. Specific rabbit polyclonal and mouse monoclonal antibodies against esterase D were prepared and recognized either denatured or native human esterase D protein. Moreover, the polyclonal antibodies immunoprecipitated a polypeptide with a molecular mass of about 33-34 kDa from various cell lines of different mammalian species, indicating that the esterase D protein is highly conserved. The highest levels of this enzyme were found in liver and kidney. Furthermore, the expression of esterase D was enhanced 3-fold in a promonocytic cell line treated with phenobarbital but not with phorbol myristate acetate, suggesting that esterase D may have a role in detoxification. The availability of the homogeneous protein and its specific antibodies allows for cloning of the esterase D gene and facilitates studies of retinoblastomas.
The avian beta-adrenergic receptor: primary structure and membrane topology.
Authors: Yarden Y , Rodriguez H , Wong S K , Brandt D R , May D C , Burnier J , Harkins R N , Chen E Y , Ramachandran J , Ullrich A
Published: May 2006

Partial amino acid sequence information allowed the isolation of cDNA clones encoding the turkey erythrocyte beta-adrenergic receptor. Antisera raised against synthetic peptides encoded by the cDNA crossreacted with the purified receptor and appropriate tryptic fragments, confirming the identity of the cDNA. The receptor is composed of 483 amino acids and has a molecular mass of 54 kDa. Its sequence suggests that it is arranged predominantly in seven membrane-spanning sequences and a long cytoplasmic carboxyl-terminal domain. The extracellular amino-terminal domain contains a consensus sequence for N-glycosylation. The beta-adrenergic receptor displays overall structural similarity and weak sequence homology with rhodopsin. Because both proteins act by regulating GTP-binding proteins, a compact structure based on seven membrane-spanning regions may be a general model for receptors that act on G proteins.
Human systemic lupus erythematosus sera contain antibodies against cell-surface protein(s) that share(s) epitope(s) with DNA.
Authors: Jacob L , Lety M A , Bach J F , Louvard D
Published: May 2006

In previous work, a murine monoclonal anti-DNA antibody (PME77) with specificity for double-stranded DNA has been found to bind five polypeptides (34, 33, 17, 16, and 14 kDa) that are expressed at the surface of several human cell types involved in lupus pathogenesis. To determine more precisely the nature of the antigens recognized by the PME77 monoclonal antibody, and to release cell-surface-accessible fragments, we used a mild, controlled elastase treatment. We isolated several of these polypeptides by immunoaffinity chromatography. A polyclonal antibody was prepared by immunizing a rabbit with a mixture of these polypeptides (17, 16, and 14 kDa) adsorbed on nitrocellulose. This antibody was shown to react with 17-, 16-, and 14-kDa polypeptides. This antibody does not bind to double-stranded DNA, suggesting that most of the immunogenic determinants of these polypeptides are not shared by double-stranded DNA. Of six human systemic lupus erythematosus sera tested, all contained antibodies that recognized this cell-surface protein(s) and crossreacted with double-stranded DNA. We suggest that this protein(s) be called LAMP [lupus-associated membrane protein(s)].
Proenkephalin is processed in a projection-specific manner in the rat central nervous system.
Authors: White J D , Gall C M , McKelvy J F
Published: May 2006

The biosynthesis and posttranslational proteolytic processing of proenkephalin was studied in three projection systems in the rat central nervous system--the caudate-putamen to the globus pallidus, the paraventricular nucleus of the hypothalamus to the median eminence, and the mossy fiber system of the granule cells of the hippocampus. By using the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification coupled with chemical modification, the biosynthesis of six radiolabeled [Met]enkephalin-containing peptides--[Met5]enkephalin, [Met5,Arg6,Gly7,Leu8]enkephalin, [Met5,Arg6,Phe7]enkephalin, metorphamide, peptide E, and BAM 18P--was followed. In each projection system, radiolabeled enkephalins were purified to constant radiochemical specific activity. However, the posttranslational processing of proenkephalin was found to differ between these three systems, as judged by the relative ratio of these peptides. These findings imply that specific, different physiologies and behaviors may be elicited by the enkephalins based upon the specific [Met]enkephalin-containing peptides that are cleaved from proenkephalin and released in synaptic terminal fields.
Thermal-mechanical fluctuations enhance repulsion between bimolecular layers.
Authors: Evans E A , Parsegian V A
Published: May 2006

Beginning with the free energy potential for long-range interactions between parallel flat sheets and the mechanical energy of bending those sheets, we have derived an upper bound for repulsion enhanced by thermal undulations of sheets in a multilamellar array. Through a self-consistent (mean-field) potential, we cover the full range of layer separations from close-in to far-apart, where the enhanced repulsion approaches the weak steric interaction derived previously by Helfrich [Helfrich, W. (1978) Z. Naturforsch. 33a, 305-315]. We have examined the effect of the fluctuation-enhanced repulsion in experimental studies of osmotic (and mechanical) compression of multilamellar lipid arrays, mechanical compression of bilayers immobilized on mica substrates, and controlled adhesion of giant bilayer membrane vesicles.
Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis.
Authors: Saluz H P , Jiricny J , Jost J P
Published: May 2006

Genomic sequencing was used to study the extent of cytosine methylation of four CpG sites within the regulatory region of the estradiol-inducible avian vitellogenin II gene. Three of these sites, two of which lie within the estradiol-receptor binding site and one in a short stretch of alternating purines and pyrimidines, were initially fully methylated. Analysis of DNA isolated from liver nuclei revealed that hormone treatment of immature White Leghorn roosters resulted in a demethylation of these sites, which occurred initially in only one DNA strand. This demethylation correlated well with the induction of vitellogenin mRNA synthesis. The demethylation of the complementary DNA strand lagged approximately equal to 24 hr behind. The fourth CpG, located within an overlapping glucocorticoid-receptor binding site, was already hemimethylated at the onset of the experiment. The demethylation of this site also occurred with kinetics similar to the rate of vitellogenin mRNA synthesis. All four CpGs remained demethylated even after cessation of gene transcription. A comparison of the methylation state of these four sites in DNA from different tissues demonstrated a clear dependence of the demethylation on estradiol. Our results suggest that this hormone-dependent event occurs via an active pathway through excision repair and/or enzymatic demethylation.
Gene-culture coevolution of complex social behavior: human altruism and mate choice.
Authors: Rushton J P , Littlefield C H , Lumsden C J
Published: May 2006

The hypothesis is examined that genes bias the development of complex social behavior in one direction over alternatives. Studies of altruism and political attitudes in twins estimate that approximately 50% of the variance is associated with direct genetic inheritance, virtually 0% with the twin's common family environment, and the remainder with each twin's specific environment. Studies of human marriages show that spouses choose each other on the basis of similarity, assorting on the most genetically influenced of a set of homogeneous attributes. These data imply a genetic canalization of social influences such that, within the constraints allowed by the total spectrum of cultural alternatives, people create environments maximally compatible with their genotypes.
Assignment of the human and mouse prion protein genes to homologous chromosomes.
Authors: Sparkes R S , Simon M , Cohn V H , Fournier R E , Lem J , Klisak I , Heinzmann C , Blatt C , Lucero M , Mohandas T
Published: May 2006

Purified preparations of scrapie prions contain one major macromolecule, designated prion protein (PrP). Genes encoding PrP are found in normal animals and humans but not within the infectious particles. The PrP gene was assigned to human chromosome 20 and the corresponding mouse chromosome 2 using somatic cell hybrids. In situ hybridization studies mapped the human PrP gene to band 20p12----pter. Our results should lead to studies of genetic loci syntenic with the PrP gene, which may play a role in the pathogenesis of prion diseases or other degenerative neurologic disorders.
Glucocerebrosidase processing in normal fibroblasts and in fibroblasts from patients with type I, type II, and type III Gaucher disease.
Authors: Beutler E , Kuhl W
Published: May 2006

Fibroblasts from normal subjects and patients with the three types of Gaucher disease were labeled with [3H]leucine. Glucocerebrosidase antigen was immunoprecipitated using affinity-purified Sepharose-bound antibody. Normal cells initially formed a 60-kDa polypeptide antigen that was gradually replaced by a broad band of antigen averaging 63 kDa. This position corresponds with that of mature fibroblast and placental enzyme. Processing of glucocerebrosidase in six unrelated patients with type I Gaucher disease and one patient with type III Gaucher disease was exactly the same as normal. In contrast, three patients with the severe infantile (type II) form of the disease manifested a very unstable enzyme; the 60-kDa band appeared transiently and the mature 63-kDa band was never seen. These results indicate that type II Gaucher disease may well be distinguishable from type I disease by virtue of the very unstable enzyme precursor. Contrary to some earlier reports, processing of glucocerebrosidase in type I disease appears to be entirely normal.
Somatostatin selectively enhances acetylcholine-induced excitations in rat hippocampus and cortex.
Authors: Mancillas J R , Siggins G R , Bloom F E
Published: May 2006

The neuronal effects of somatostatin-14 (SS-14) and its influence on responses to acetylcholine (AcCho) were studied in vivo in the rat parietal cortex and dorsal hippocampus, using single-unit recording and microiontophoresis. SS-14 inhibited spontaneous firing of nearly all cells tested, while AcCho facilitated their firing. In contrast to its direct slowing effect, sustained iontophoretic application of SS-14 enhanced AcCho-induced excitations in 78% of all cells tested. This AcCho-enhancing effect of SS-14 was dose dependent. SS-14 did not enhance the responsiveness to pulses of the excitatory amino acid glutamate. Neurons tonically driven by iontophoretic currents of AcCho responded to concurrent pulses of SS-14 with an increase in firing. Thus, iontophoretic application of SS-14 can produce qualitatively different effects on the spontaneous activity of its target cells depending on the simultaneous effects of other chemical messengers. These condition-dependent interactions may explain the diverse neuronal effects of SS-14 reported in the literature.
Expression of the major glycoprotein G of human respiratory syncytial virus from recombinant vaccinia virus vectors.
Authors: Ball L A , Young K K , Anderson K , Collins P L , Wertz G W
Published: May 2006

The major glycoprotein, G, of human respiratory syncytial (RS) virus is a Mr 84,000-90,000 species that has about 60% of its mass contributed by carbohydrate, most of which is in the form of O-linked oligosaccharides. The G protein contains neither a hydrophobic N-terminal signal sequence nor a hydrophobic C-terminal anchor region. Instead, its amino acid sequence reveals only one region with significant hydrophobic character, which is between residues 38 and 66. In order to study the synthesis, processing, and functions of this unusual viral glycoprotein, full-length cDNA copies of the G protein mRNA were inserted into the DNA genome of vaccinia virus (VV) in a position that was adjacent to a strong VV promoter and within the VV gene for thymidine kinase (TK). The resulting TK- recombinant viruses were selected, plaque-purified, and characterized by Southern blot analysis of restriction enzyme digests of the viral DNA. Recombinant RNA transcripts that contained both G-specific and VV-specific sequences accumulated in cells infected with recombinant viruses having the G protein gene in the positive orientation. The translation product of these transcripts in infected cells was a Mr 84,000-90,000 glycoprotein that was indistinguishable from authentic RS virus G protein. It could be detected in cell lysates after metabolic labeling with [3H]glucosamine and was immunoprecipitated by anti-RS-virus antiserum. Immunofluorescence studies showed that the G protein accumulated intracellularly with the perinuclear distribution that is characteristic of newly synthesized glycoproteins. Furthermore, the protein was also clearly detectable on the surface of recombinant-infected cells, showing that it was transported to and inserted into the plasma membrane.
The gene for the human immune interferon receptor is located on chromosome 6.
Authors: Rashidbaigi A , Langer J A , Jung V , Jones C , Morse H G , Tischfield J A , Trill J J , Kung H F , Pestka S
Published: May 2006

When 32P-labeled human recombinant immune interferon gamma (Hu-[32P]IFN-gamma) is crosslinked to human cells with disuccinimidyl suberate, a complex with a molecular size of approximately equal to 117,000 Da was identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The formation of this complex is inhibited when the binding is performed in the presence of excess unlabeled Hu-IFN-gamma. The specific formation of the 117,000-Da complex is not observed in mouse L cells or Chinese hamster ovary cells. This complex shows all of the criteria that identify it as the Hu-IFN-gamma receptor or its binding subunit. The same complex can be formed following binding and covalent crosslinking of Hu-[32P]IFN-gamma to some hamster-human or mouse-human somatic cell hybrids. The presence of human chromosome 6 in the hybrids is necessary and sufficient for the formation of this complex. More specifically, the long arm of chromosome 6 seems sufficient. Therefore, we have localized the gene for the Hu-IFN-gamma receptor (or its binding subunit) to the long arm of human chromosome 6. The presence of this chromosome in the somatic cell hybrids is not adequate, however, to confer antiviral resistance to the hybrids in the presence of Hu-IFN-gamma.
Chromosomal localization of the human apolipoprotein B gene and detection of homologous RNA in monkey intestine.
Authors: Deeb S S , Disteche C , Motulsky A G , Lebo R V , Kan Y W
Published: May 2006

A cDNA clone of the human apolipoprotein B-100 was used as a hybridization probe to detect homologous sequences in both flow-sorted and in situ metaphase chromosomes. The results indicate that the gene encoding this protein is on the distal end of the short arm of chromosome 2 (2p23-2p24). RNA isolated from monkey small intestine contained sequences (6.5 and 18 kilobases) homologous to the cDNA of apolipoprotein B-100. These results are consistent with the hypothesis that one gene codes for both the intestinal (B-48) and the hepatic (B-100) forms.
Recombinant human tumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigens in vascular endothelial cells and dermal fibroblasts in vitro.
Authors: Collins T , Lapierre L A , Fiers W , Strominger J L , Pober J S
Published: May 2006

Recombinant human tumor necrosis factor (TNF), purified to greater than 99% homogeneity, increases surface expression of class I major histocompatibility complex (MHC) antigens to a maximum of 9-fold on cultured human endothelial cells (HEC) and human dermal fibroblasts (HDF). The increase is concentration dependent (peak 20-100 units/ml) and time dependent (nearly maximal by 4 days); expression remains elevated in the continued presence of TNF and requires greater than 7 days to return to basal levels upon TNF withdrawal. The increase in surface expression appears to result from increases in steady-state mRNA levels for the class I antigens, although the increase in mRNA is proportionately greater than for surface expression. No surface expression of or mRNA for class II MHC antigens is detectable in either control or TNF-treated HEC or HDF. These effects are similar to those produced by leukocyte or fibroblast (type I) interferons (IFNs). The protein synthesis inhibitor cycloheximide (CHX), when added coincidentally with type I IFNs, leads to superinduction of mRNA for class I MHC antigens and, unexpectedly, leads to the appearance of mRNA for class II MHC antigens. CHX has no effect by itself upon mRNA levels for class I or class II MHC antigens, nor does it modulate the increases in mRNA produced by immune (type II) IFN. Most interesting, CHX blocks the increase in mRNA for class I MHC antigens induced by TNF. Thus TNF appears to act on MHC gene expression through a newly synthesized protein intermediate. Our results provide direct evidence that TNF can modulate gene expression in normal (untransformed) cell types and contribute to understanding the complex nature of MHC gene regulation. Finally, they suggest that TNF may act in vivo as an immunoregulatory molecule.