bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of apotransferrin to K562 cells: explanation of the transferrin cycle.
Authors: Klausner R D , Ashwell G , van Renswoude J , Harford J B , Bridges K R
Published: May 2006

The binding of apotransferrin to the transferrin receptor on the surface of human leukemic K562 cells was found to be significantly less tight than that of the holoprotein, diferric transferrin. The finding that both ligands displayed linear Scatchard plots with similar receptor number (approximately equal to 150,000 per cell) and mutually inhibit each other's binding suggested that they bind to the same receptor. Both the dissociation and association rate of apotransferrin were markedly increased (28-fold and 15-fold, respectively) at pH 7.2 compared to pH 4.8. Using the values of these binding parameters, we propose a mechanism to account for the recycling of transferrin subsequent to internalization and residence within an acidic nonlysosomal organelle where iron is removed.
Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917.
Authors: Youngman P J , Perkins J B , Losick R
Published: May 2006

The Streptococcus faecalis transposon Tn917 was introduced into Bacillus subtilis by transformation of competent cells with the plasmid pAM alpha 1::Tn917 and was tested for transposition activity by selection for insertions into the temperate phage SP beta. Insertions were obtained at a frequency indicating relatively efficient movement of the element, and Southern hybridization analysis of a particular insertion confirmed it to be the result of a genuine transposition event. A restriction fragment from pAM alpha 1::Tn917 containing the transposon sequences was ligated into a temperature-sensitive plasmid (pBD95), and transpositions into the B. subtilis chromosome were selected by requiring the transposon drug resistance to be maintained at temperatures nonpermissive for plasmid replication. Insertions have been recovered at many chromosomal sites, including ones that produced auxotrophy of different kinds and ones that produced various different sporulation-defective phenotypes, indicating good prospects for the use of Tn917 as a tool for insertional mutagenesis in B. subtilis.
Role of short regions of homology in intermolecular illegitimate recombination events.
Authors: Marvo S L , King S R , Jaskunas S R
Published: May 2006

The structures of three recombinants between bacteriophage lambda DNA and plasmid pBR322 that were generated in a recA derivative of Escherichia coli are described. Each resulted from two illegitimate recombination events that resulted in the substitution of part of the lambda genome by part of the plasmid genome. The nucleotide sequences at the six lambda-plasmid junctions were determined and compared with the sequences of the lambda and plasmid genomes before recombination. Each recombination occurred at a short region of homology in the two genomes, and other short regions of homology were found near some of the junctions. The structures of these junctions are similar to those resulting from illegitimate recombination in animal cells. A model to explain how these multiple illegitimate recombination events could result from a cascade of DNA gyrase-catalyzed recombinations is discussed.
Isolation and expression of an altered mouse dihydrofolate reductase cDNA.
Authors: Simonsen C C , Levinson A D
Published: May 2006

We have constructed a cDNA library from a murine cell line expressing high levels of a dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) that displays an abnormally low affinity for methotrexate. From this library we have isolated a cDNA clone similar to, but distinguishable from, a cDNA clone previously demonstrated to encode the wild-type enzyme. Analysis of the nucleotide sequence of this cDNA clone allows us to predict that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an arginine for a leucine residue in a region of the polypeptide thought to form a hydrophobic pocket essential for inhibitor binding. To confirm that this substitution was responsible for the altered properties of the enzyme, we genetically localized the region of the cDNA that specified resistance to methotrexate by in vitro recombination. These results reveal that a single nucleotide change in the codon specifying amino acid 22 of the enzyme was sufficient to alter the methotrexate sensitivity of the enzyme. We demonstrate that this altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase.
Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro.
Authors: Gross J L , Moscatelli D , Rifkin D B
Published: May 2006

Bovine capillary endothelial (BCE) cells produce increased amounts of the proteases plasminogen activator (PA) and latent collagenase when cultured in the presence of the following preparations which are known to contain angiogenic activities: bovine retinal extract, mouse adipocyte conditioned medium, and human hepatoma cell lysate. These preparations stimulated both BCE cell PA and collagenase activities in a dose-dependent manner. Both activities were increased to about the same level by these preparations as by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. Mitogens that are not angiogenic, such as insulin, epidermal and fibroblast growth factors, and endothelial cell growth supplement, had no effect on BCE cell PA and collagenase activities. Two of the angiogenic preparations (retinal extract and mouse adipocyte-conditioned medium) had no effect on PA activity in endothelial cells derived from bovine aortae (BAE cells). The angiogenic preparations had little (human hepatoma cell lysate, mouse adipocyte-conditioned medium) or no (bovine retinal extract) effect on BAE cell collagenase activities. In the bovine system, the induction of high levels of both PA and collagenase activities by angiogenic preparations is limited to capillary endothelial cells.
Transferrin as a fetal growth factor: acquisition of responsiveness related to embryonic induction.
Authors: Ekblom P , Thesleff I , Saxén L , Miettinen A , Timpl R
Published: May 2006

Differentiation of the metanephric mesenchyme, which is triggered by an inductive tissue interaction, has been shown to proceed in a chemically defined medium containing transferrin. Here, we report that neither transferrin-depleted serum nor a chemically defined medium devoid of transferrin promote differentiation and that activity can be restored by the addition of transferrin. It thus appears that we have identified the serum factor required for kidney differentiation. Transferrin seems to affect differentiation by stimulating cell proliferation. We show by using an organ-culture model system that only mesenchymes induced to differentiate by the 24-hr tissue interaction respond to transferrin by proliferation and differentiation, whereas uninduced mesenchymes remain unresponsive. The inductor tissue used is not responsive to transferrin. Thus, the data suggest that the short-range cell-mediated tissue interaction acts by making the nephrogenic mesenchyme responsive to the long-range mediator, which is transferrin. Transferrin is suggested to be an important circulating growth factor required for proliferation during embryogenesis.
Characterization of a teleost gonadotropin-releasing hormone.
Authors: Sherwood N , Eiden L , Brownstein M , Spiess J , Rivier J , Vale W
Published: May 2006

A peptide that is recognized by certain antibodies raised against mammalian gonadotropin-releasing hormone has been purified from extracts of salmon brains by gel filtration and high-performance liquid chromatography. The primary structure of this 10-residue peptide is less than Glu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2. This represents a difference of two amino acids between salmon and mammalian gonadotropin-releasing hormone and demonstrates that most of the molecule has been conserved during evolution. The synthetic form of salmon gonadotropin-releasing hormone is less potent than is mammalian gonadotropin-releasing hormone on mammalian cells and is biologically active in salmon.
Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D.
Authors: Paoletti E , Lipinskas B R , Samsonoff C , Mercer S , Panicali D
Published: May 2006

Potential live vaccines using recombinant vaccinia viruses have been constructed for both hepatitis B and herpes simplex. These recombinant vaccinia viruses express cloned genes of the hepatitis B virus surface antigen (HBsAg) or the glycoprotein D from herpes simplex virus (HSV-gD). The HBsAg synthesized in vitro under the regulation of vaccinia virus is secreted from infected cells as a particle of approximately equal to 22 nm diameter with a density of 1.2 g/ml as determined on CsCl gradients. Inoculation of rabbits with the recombinant vaccinia virus that expresses the HBsAg elicits the production of high-titered antibodies. Synthesis of the HSV-gD was detected in tissue culture by radioimmunoassay on unfixed cells, suggesting that the HSV-gD synthesized by the recombinant vaccinia virus is membrane associated. Inoculation of rabbits with the recombinant vaccinia virus expressing HSV-gD resulted in the production of antibodies that reacted with authentic HSV-gD as detected by radioimmunoassay. Furthermore, the anti-serum was shown by plaque-reduction assay to neutralize the infectivity of herpes simplex virus. Immunization of mice with the vaccinia recombinant expressing HSV-gD gave complete protection on subsequent challenge with lethal doses of live herpes simplex virus.
Distribution of two selenonucleosides among the selenium-containing tRNAs from
Methanococcus vannielii
Authors: Ching Wei-Mei , Wittwer Arthur J. , Tsai Lin , Stadtman Thressa C.
Published: May 2006

Naturally occurring seleno-tRNAs from
Methanococcus vannielii
account for 13-20% of the total tRNA population. Two different selenium-modified nucleosides were detected in these seleno-tRNAs. Of the total selenium incorporated, about 60% is present in 5-methylaminomethyl-2-selenouridine, and the other 40% occurs in a second selenonucleoside with spectral characteristics typical of a derivative of 2-selenouridine. The 5-methylaminomethyl-2-selenouridine was found in the seleno-tRNA species present in the early fractions of a linear salt gradient elution profile from a reversed-phase chromatographic system 5(RPC-5) column, whereas the second selenonucleoside occurred in the tRNA species eluted late in the profile.
Isolation of transforming sequences of two human lung carcinomas: structural and functional analysis of the activated c-K-ras oncogenes.
Authors: Nakano H , Yamamoto F , Neville C , Evans D , Mizuno T , Perucho M
Published: May 2006

Human lung tumors PR310 and PR371 maintained in nude mice contain activated c-K-ras oncogenes detectable by the ability of their DNAs to induce the morphological transformation of NIH 3T3 mouse fibroblasts. Using phage libraries constructed with DNA from NIH 3T3 mouse fibroblast transformants, we have isolated human sequences that span greater than 40 kilobase pairs of the c-K-ras oncogene. Based on the conservation of these human sequences in mouse fibroblast transformants, we conclude that the transforming ability of the oncogene activated in these tumors resides within a 43- to 46-kilobase-pair DNA region. No clear differences were observed between the structures of the PR310 and PR371 cloned oncogene sequences. Nucleotide sequence analysis in concert with DNA transfection experiments suggests that the PR371 oncogene has been activated by a single base change in the first exon, which results in the substitution of cysteine for glycine in position 12 of the predicted amino acid sequence. The genetic alteration responsible for the transforming activity of the PR310 oncogene, however, does not reside in the first exon. These results indicate that the activation of the c-K-ras oncogene in human lung cancer can occur by different mutational events.
Characterization of genomic clones specifying rabbit alpha- and beta-ventricular myosin heavy chains.
Authors: Friedman D J , Umeda P K , Sinha A M , Hsu H J , Jakovcic S , Rabinowitz M
Published: May 2006

We have isolated gene sequences coding for the alpha- and beta-myosin heavy chains (HC) of rabbit ventricular muscle. A rabbit genomic library was screened with previously characterized cDNA clones specifying part of the light meromyosin and the entire subfragment 2 portion of alpha- and beta-myosin HCs, as well as with a clone containing the 3' nontranslated sequences of the alpha-myosin HC mRNA. Seven strongly hybridizing clones were analyzed in detail. One genomic clone encoded all of the 3' nontranslated sequences of an alpha-cDNA clone and, therefore, contained the 3' end of the alpha-myosin HC gene. Electron microscopic heteroduplex analysis and DNA sequence analysis showed that this clone overlapped a second genomic clone providing more than 25 kilobase pairs of the alpha-myosin HC gene. The exons within this region corresponded to approximately equal to 85% of the mRNA and were separated by at least 28 introns. A clone for the beta-myosin HC gene was also identified by Southern blot hybridization, by heteroduplex mapping, and by comparing the DNA sequence of a subfragment 2 exon to sequences of the alpha- and beta-cDNA clones. The introns of the alpha- and beta-myosin HC genes were in the same position but showed marked variation in length. These results conclusively showed that the alpha- and beta-myosin HCs are products of separate genes.
Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.
Authors: Taft W C , DeLorenzo R J
Published: May 2006

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.
Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA.
Authors: Kucherlapati R S , Eves E M , Song K Y , Morse B S , Smithies O
Published: May 2006

We have used the eukaryotic-prokaryotic shuttle vector pSV2Neo to demonstrate that cultured mammalian somatic cells have the enzymatic machinery to mediate homologous recombination and that the frequency of this recombination can be enhanced by pretreatment of the input DNA. Two nonoverlapping deletion mutants of pSV2Neo were constructed, each affecting the bacterial aminoglycoside 3'-phosphorylase gene (the neo gene), which confers resistance to aminoglycoside antibiotics on bacteria and resistance to the antibiotic G418 on mammalian cells. Mammalian cells transfected with either deletion plasmid alone yield no G418 -resistant colonies. Cells cotransfected with both deletion plasmids yield G418 -resistant colonies with high frequency. We show that these resistant colonies result from recombination involving homologous crossing-over or gene conversion between the deletion plasmids by rescuing from the resistant cells both types of reciprocal recombinant, full-length plasmids, and doubly deleted plasmids. Cutting one of the input plasmids to generate a double-stranded gap in the neo gene considerably enhances the frequency of homologous recombination within the gene. This suggests that targeting exogenous DNA to specific sites in mammalian chromosomes could be facilitated by suitable pretreatment of the DNA.
Autoantibodies to glucosylated proteins in the plasma of patients with diabetes mellitus.
Authors: Witztum J L , Steinbrecher U P , Kesaniemi Y A , Fisher M
Published: May 2006

Nonenzymatic glucosylation interferes with recognition of low density lipoprotein (LDL) by its receptor and markedly decreases the rate of plasma clearance of glucosylated LDL, both in experimental animals and in normal human subjects. However, in selected diabetic subjects we have observed a paradoxical increase in the clearance of glucosylated LDL, suggesting the possibility of immune-mediated clearance. Immunoassay demonstrated antibodies specific for glucosylated LDL in the preinjection plasma of each of four such diabetic subjects studied. These antibodies cross-react with other glucosylated proteins and recognize specifically the glucosylated lysine epitope--i.e., glucitollysine . These data suggest that nonenzymatic glucosylation of plasma or structural proteins may render them immunogenic and result in production of autoantibodies that recognize not only the particular immunogen but also many other glucosylated proteins, including glucosylated tissue proteins. These findings may be relevant to the increased prevalence of immune complexes in plasma of diabetic subjects and the late complications of diabetes mellitus.
Periodic structures in lipid monolayer phase transitions
Authors: McConnell Harden M. , Tamm Lukas K. , Weis Robert M.
Published: May 2006

Periodic patterns are observed when supported lipid monolayers doped with low concentrations of fluorescent lipid probes are observed with epi-fluorescence microscopy. Monolayers of dipalmitoyl phosphatidylcholine were examined on air-water interfaces and also on alkylated glass coverslips. The patterns are formed by periodic arrays of solid-phase lipid domains in equilibrium with fluid-phase lipid under specified conditions of temperature and two-dimensional lipid pressure. Electrostatic forces may stabilize the periodic ordering of the solid domains.
NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD.
Authors: Carroll S F , Collier R J
Published: May 2006

We showed earlier that exposing mixtures of NAD and diphtheria toxin fragment A to ultraviolet radiation (253.7 nm) induced the formation of covalently linked protein-ligand photoproducts. Here we report that when [carbonyl-14C]NAD was employed in such procedures, the efficiency of labeling of the protein approached 1 mol/mol, and at least 94% of the incorporated label was associated with a single residue, glutamic acid at position 148. Fragment A photolabeled in this manner was enzymically inactive. The efficiency of photolabeling was much lower (less than 0.2 mol/mol) when NAD radiolabeled in either the adenine moiety or the adenylate phosphate was used, and the label was attached to different site(s) within fragment A. Efficient photochemical transfer of label from [carbonyl-14C]NAD occurred under identical conditions with the nucleotide-free form of whole diphtheria toxin, CRM-45, or activated exotoxin A from Pseudomonas aeruginosa, but not with nucleotide-bound diphtheria toxin, CRM-197, native exotoxin A, or any of several NAD-linked dehydrogenases. On the basis of these and other results we suggest that part or all of the nicotinamide moiety of NAD is efficiently transferred to glutamate-148 of fragment A under the influence of ultraviolet irradiation and that this residue is located within the nicotinamide subsite. This location implies that glutamate-148 is at or near the catalytic center of the toxin. Our data provide direct evidence for the location of the NAD site in an ADP-ribosylating toxin and demonstrate highly efficient and specific photolabeling by [carbonyl-14C]NAD.
Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin.
Authors: Strålfors P , Björgell P , Belfrage P
Published: May 2006

In isolated adipocytes, fast-acting lipolytic hormones and insulin have been shown previously to control lipolysis by regulating the activity of hormone-sensitive lipase, the rate-limiting enzyme, through an increase or decrease, respectively, of the extent of phosphorylation of the enzyme. Here, we demonstrate that exposure to lipolytic hormones (corticotropin, noradrenaline) led to phosphorylation at two sites on the Mr 84,000 lipase subunit. One, designated "basal site," was phosphorylated also in the absence of any hormonal stimulation, its phosphorylation apparently not being influenced by hormones. The second, designated "regulatory site," was identical to that phosphorylated by cyclic AMP-dependent protein kinase on the isolated lipase. The regulatory site was not appreciably phosphorylated in the absence of hormones, but exposure of the cells to noradrenaline increased its phosphorylation extent to that of the basal site. Insulin or the beta-adrenergic antagonist propranolol decreased the extent of phosphorylation of the regulatory site to the low level before stimulation, apparently without effect on the basal site. Phosphoserine was the only phosphorylated amino acid residue at both sites. Limited proteolytic digestion indicated that the two sites were separated by less than about 170 amino acid residues. Thus, control of adipose tissue lipolysis by fast-acting lipolytic hormones and by insulin is exerted through the regulation of the phosphorylation state of a single phosphoserine residue in the hormone-sensitive lipase.
Cell swelling increases a barium-inhibitable potassium conductance in the basolateral membrane of Necturus small intestine.
Authors: Lau K R , Hudson R L , Schultz S G
Published: May 2006

Previous studies have shown that, immediately after the addition of galactose or alanine to the solution bathing the mucosal surface of Necturus small intestine, there is a rapid depolarization of the electrical potential difference across the mucosal membrane (psi mc). This is followed by a repolarization of psi mc that is paralleled by an increase in the ratio of the effective resistance of the mucosal membrane to that of the basolateral membrane (rm/rs); the latter was shown to be, at least in part, due to a marked increase in the conductance of the basolateral membrane. We now report the following. (i) Exposure of this epithelium to a 12% hypotonic solution results in a hyperpolarization of psi mc and an increase in rm/rs. These effects are blocked by metabolic inhibitors and by the presence of 5 mM Ba2+ in the bathing solution; indeed, in the presence of Ba2+, psi mc depolarizes and rm/rs decreases to low values. (ii) Addition of 15 mM galactose to the mucosal solution when the serosal solution alone contains 5 mM Ba2+ results in a depolarization of psi mc but no subsequent repolarization of psi mc or increase in rm/rs; however, psi mc repolarizes and rm/rs increases when Ba2+ is subsequently removed from the serosal bathing solution. We conclude that (i) the basolateral membrane normally possesses a Ba2+-inhibitable K conductance, which appears to be reduced in the presence of metabolic inhibitors; (ii) after exposure of the tissue to a hypotonic solution or the addition of galactose to the mucosal solution, this conductance increases; and (iii) these responses can be blocked by metabolic inhibitors. These findings suggest that the delayed response of this tissue to the addition of sugars or amino acids to the mucosal solution may be the result of cell swelling resulting from the intracellular accumulation of these solutes in osmotically active forms.
Desulfovibrio vulgaris hydrogenase: a nonheme iron enzyme lacking nickel that exhibits anomalous EPR and Mössbauer spectra.
Authors: Huynh B H , Czechowski M H , Krüger H J , DerVartanian D V , Peck H D , LeGall J
Published: May 2006

A purification procedure for the periplasmic hydrogenase from Desulfovibrio vulgaris ( Hildenborough , National Collection of Industrial Bacteria 8303) is reported. The purified hydrogenase has a specific activity of 4800 units per mg of protein. Plasma emission studies reveal that this highly active hydrogenase is free of nickel and contains 11 (+/- 1) nonheme iron atoms per molecule. A combined EPR and Mössbauer study indicates that the majority of the iron atoms are bound in the form of iron- sulfur clusters. Two ferredoxin-type [4Fe-4S] clusters have been identified that exhibit normal EPR and Mössbauer parameters; however, no trace of 3Fe cluster is detected by the Mössbauer measurement. In the presence of oxidants, cytochrome c3, and CO, anomalous EPR and Mössbauer spectra indicative of atypical nonheme iron centers are observed.
Indole-3-acetic acid sensitization of phytochrome-controlled growth of coleoptile sections
Authors: Shinkle James R. , Briggs Winslow R.
Published: May 2006

Addition of 6 μM indole-3-acetic acid (IAA) to incubation buffer increases the sensitivity of coleoptile sections cut from dark-grown
Avena sativa
L. cv. Lodi to red light by a factor of 10,000, relative to the response in the absence of added IAA, without changing the maximum amount of light-induced growth. From 0.03 to 4 μM IAA sections show at least a 100-fold increase in sensitivity to red light relative to the response in the absence of added IAA. In this IAA concentration range, the light-induced increase in elongation shows two phases of response to red-light fluence, which are separated by a plateau. The biphasic fluence-response curve is also characteristic of the red-light-induced stimulation of coleoptile growth in intact dark-grown seedlings. The effect of IAA on the sensitivity of the phytochrome-mediated growth response appears to be on some step in the transduction of the phytochrome signal, rather than on the growth response itself.
Immunosuppression in vitro by a metabolite of a human pathogenic fungus.
Authors: Müllbacher A , Eichner R D
Published: May 2006

Gliotoxin , a metabolite of Aspergillus fumigatus, inhibits phagocytosis of macrophages at concentrations of 20-50 ng/ml. Pretreatment of stimulator cells in mixed lymphocyte cultures with gliotoxin (100 ng/ml) abrogates induction of alloreactive cytotoxic T cells. The presence of gliotoxin 48 hr after initiation of cytotoxic T-cell induction has no effect. Inhibition of cytotoxic T-cell induction by gliotoxin at low concentrations, acting on the stimulator cells, can be overridden by concanavalin A-activated cell supernatants. Gliotoxin does not induce immediate cell-surface antigen modification on target cells. The possible role of gliotoxin in the etiology of A. fumigatus-related diseases is discussed.
Serum alpha-fetoprotein levels in human disease: perspective from a highly specific monoclonal radioimmunoassay.
Authors: Bellet D H , Wands J R , Isselbacher K J , Bohuon C
Published: May 2006

A rapid multisite radioimmunoassay for measurement of human alpha-fetoprotein (AFP) that uses two high-affinity monoclonal antibodies directed against distinct and separate determinants on the protein was developed and designated M-RIA. The sensitivity of the "simultaneous-sandwich" M-RIA is approximately equal to 0.5 ng/ml of serum after a 1-hr incubation period. Serum AFP levels have been measured in 1747 individuals with hepatocellular carcinoma (HCC), acute and chronic hepatitis B virus infection, chronic hepatitis B surface antigen (HBsAg)-carrier states, cirrhosis, other malignant tumors, and normal and disease controls to determine the specificity of the assay. Eighty percent (68/85) of patients with HBsAg-positive HCC had AFP levels of greater than 200 ng/ml (range, 260 to greater than 200,000 ng/ml). In contrast, all 450 normal subjects and 477 chronic HBsAg-positive carriers had levels of less than 20 ng/ml. More importantly, in acute and chronic hepatitis B, cirrhosis, and other malignant tumors and in the remaining disease controls, AFP levels were less than 20 ng/ml in 99.3% of the subjects, the great majority (greater than 96%) being less than 5 ng/ml. Indeed only two of 1635 individuals, one with acute hepatitis and the other with carcinoma of the esophagus had AFP levels of greater than 100 ng/ml. These observations are at variance with previous studies with conventional polyvalent RIAs of AFP levels of greater than 20 ng/ml in approximately equal to 40% of acute and chronic hepatitis and in 30% of cirrhosis. This striking specificity of the M-RIA is probably due in part to recognition of epitopes unique to AFP and suggest that such an assay may be used in the detection, early identification, and monitoring of AFP-producing tumors in high-risk populations.
Neurotransmitter receptors mediate cyclic GMP formation by involvement of arachidonic acid and lipoxygenase.
Authors: Snider R M , McKinney M , Forray C , Richelson E
Published: May 2006

Evidence is presented that has led us to abandon the hypothesis that receptor-mediated cyclic GMP formation in cultured nerve cells occurs via the influx of extracellular calcium ions and an increase in the cytosolic free calcium ion concentration. While the cyclic GMP response is absolutely dependent on the presence of Ca2+, there is no increase in free intracellular Ca2+ subsequent to agonist stimulation. Instead, we have found that muscarinic or histamine H1 receptor stimulation elicits the release of arachidonic acid through a quinacrine-sensitive mechanism, possibly phospholipase A2. Inhibition of the release or metabolism of arachidonate by the lipoxygenase pathway prevents receptor-mediated cyclic GMP formation. We hypothesize that neurotransmitter receptors that mediate cyclic GMP synthesis function by releasing arachidonic acid and that an oxidative metabolite of arachidonic acid then stimulates soluble guanylate cyclase.
Tandem promoters preceding the gene for the M1 RNA component of Escherichia coli ribonuclease P.
Authors: Motamedi H , Lee Y , Schmidt F J
Published: May 2006

The nucleotide sequence of a cloned gene for the RNA component of Escherichia coli ribonuclease P, M1 RNA, is presented. The sequence determined extends 320 nucleotides upstream of the 377-base-pair (bp) structural gene and includes three sequences homologous to the consensus E. coli promoter sequence. Two nucleotides found in the M1 RNA structural gene sequence were not found in a previously determined gene sequence of another M1 RNA clone [Reed, R. E., Baer, M. F., Guerrier-Takeda, C., Donis-Keller, H. & Altman, S. (1982) Cell 30, 627-636]. In vitro transcription of supercoiled plasmid DNA containing the M1 RNA gene resulted in a major transcript arising from the strong promoter nearest to the mature M1 RNA. RNAs encoded by the M1 RNA clone in vivo were examined by S1 nuclease mapping. The results indicated that in vivo transcripts originate from all three promoters preceding the M1 RNA gene. These transcripts are apparently processed in a multistep pathway to generate the 5' end of mature M1 RNA.
Inhibition of protein synthesis stimulates the transcription of human beta-interferon genes in Chinese hamster ovary cells.
Authors: Ringold G M , Dieckmann B , Vannice J L , Trahey M , McCormick F
Published: May 2006

Using Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the human beta-interferon gene, we find that inhibitors of protein synthesis, in the absence of any other inducer, stimulate the production of interferon RNA; this effect is maintained in cells in which the plasmid sequences have been amplified 25- to 50-fold. Nuclear transcription assays show that a major effect of cycloheximide is to increase the rate of transcription of the interferon gene. This contradicts the generally accepted explanation that inhibitors of protein synthesis augment interferon production by stabilizing interferon mRNA. In addition, we have studied the effects of double stranded RNA [poly(rI) X poly(rC)] on the induction of interferon RNA in the presence and absence of cycloheximide. Our results indicate that poly(rI) X poly(rC) by itself causes a transient increase in interferon RNA; however, in the presence of cycloheximide this effect is prolonged. We do not, however, find an increase in transcription of the interferon gene(s) as an early response to poly(rI) X poly(rC). Finally, we have found that cells treated with cycloheximide or infected with Newcastle disease virus induce large amounts of a secreted 11-kDa protein. This cellular protein is not inducible by poly(rI) X poly(rC). We propose that both interferon and this 11-kDa protein belong to a family of proteins in which production is regulated in a coordinate fashion during viral inhibition of cellular protein synthesis.
Single gene for the large subunit of ribulosebisphosphate carboxylase in maize yields two differentially regulated mRNAs.
Authors: Crossland L D , Rodermel S R , Bogorad L
Published: May 2006

A second mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] has been found in the plastids of maize leaves. The 5' terminus of this mRNA was shown by S1 nuclease analysis to be 238 nucleotides upstream of the previously described large subunit mRNA [McIntosh, L., Poulsen, C. & Bogorad, L. (1980) Nature (London) 288, 556-560]. The same two mRNAs were produced in a homologous in vitro transcription system using cloned plastid DNA as a template. The ratio of the two mRNAs changes during light-induced plastid development and can be altered in the in vitro system by manipulation of transcription conditions.
Neuroblastoma cells produce transforming growth factors during exponential growth in a defined hormone-free medium.
Authors: van Zoelen E J , Twardzik D R , van Oostwaard T M , van der Saag P T , de Laat S W , Todaro G J
Published: May 2006

Mouse neuroblastoma Neuro-2A cells have been cultured in a chemically defined serum-free medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 30 nM selenite and 10 micrograms of transferrin per ml. In this medium, which does not contain any externally added polypeptide growth factor, cells proliferate rapidly with a doubling time of approximately equal to 10 hr. During exponential growth in this serum-free medium, Neuro-2A cells secrete a 15- to 20-kDa transforming growth factor with strong mitogenic action and the ability to induce anchorage-independent growth on nontransformed cells. This neuroblastoma-derived transforming growth factor (ND-TGF) is acid and heat stable but is sensitive to treatment with trypsin or dithiothreitol. However, it does not compete with epidermal growth factor (EGF) for receptor binding and does not require EGF receptors for its mitogenic activity. Experiments on the effects of EGF on ND-TGF-induced soft agar growth of normal rat kidney cells indicate that Neuro-2A cells secrete an EGF-potentiated TGF in addition to ND-TGF. It is suggested that Neuro-2A cells can proliferate in the absence of externally added growth factors as a result of autocrine production of polypeptide growth factors.
Identification of genes and gene products necessary for bacterial bioluminescence.
Authors: Engebrecht J , Silverman M
Published: May 2006

Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by complementation in trans with a series of polar transposon insertions on other plasmids. lux genes were defined by complementation of lux gene defects on pairs of plasmids in trans in E. coli. Hybrid plasmids were also used to direct the synthesis of polypeptides in the E. coli minicell system. Seven lux genes and the corresponding gene products were identified from the complementation analysis and the minicell programing experiments. These genes, in the order of their position on a linear map, and the apparent molecular weights of the gene products are luxR (27,000), luxI (25,000), luxC (53,000), luxD (33,000), luxA (40,000), luxB (38,000), and luxE (42,000). From the luminescence phenotypes of E. coli containing mutant plasmids, functions were assigned to these genes: luxA, luxB, luxC, luxD, and luxE encode enzymes for light production and luxR and luxI encode regulatory functions.
Glucocorticoid-mediated induction of alpha 1-acid glycoprotein: evidence for hormone-regulated RNA processing.
Authors: Vannice J L , Taylor J M , Ringold G M
Published: May 2006

We have studied the glucocorticoid-mediated accumulation of alpha 1-acid glycoprotein (AGP) in mRNA in HTC rat hepatoma cells. In contrast to the well-characterized primary response of mouse mammary tumor virus, in vitro transcription assays in isolated nuclei show that the rate of transcription of the AGP gene is high even in the absence of hormone. Despite the constitutive transcription of the AGP gene, no detectable AGP RNA can be found in either the cytoplasm or the nuclei of untreated cells. Previous experiments have shown that the glucocorticoid induction of AGP RNA requires ongoing protein synthesis. In conjunction with the present study, our data suggest that glucocorticoids stimulate accumulation of AGP RNA by inducing an RNA processing factor that allows production of stable transcripts.
Phorbol ester induces desensitization of adenylate cyclase and phosphorylation of the beta-adrenergic receptor in turkey erythrocytes.
Authors: Kelleher D J , Pessin J E , Ruoho A E , Johnson G L
Published: May 2006

Incubation of turkey erythrocytes with the phorbol ester phorbol 12-myristate 13-acetate (PMA) results in a dose- and time-dependent desensitization of isoproterenol-stimulated adenylate cyclase activity. Compared to controls, membranes from PMA-treated cells have an isoproterenol-stimulated adenylate cyclase activity that is decreased 20%-40%, with little effect on forskolin or fluoride activation of adenylate cyclase. No change in beta-adrenergic receptor number is observed after PMA treatment, indicating that the major effect of PMA is to uncouple receptor interactions with Ns, the stimulatory guanine nucleotide regulatory protein of adenylate cyclase. Purification of beta-adrenergic receptors from 32Pi-labeled turkey erythrocytes, incubated in the presence or absence of PMA, indicates that the phorbol ester is capable of inducing a 3-fold increase in phosphorylation of the beta-adrenergic receptor. The PMA effect is similar to the phosphorylation of the beta-adrenergic receptor during isoproterenol- and dibutyryl cAMP-induced desensitization of adenylate cyclase in turkey erythrocytes. The findings indicate that decreased receptor-Ns coupling is correlated with receptor phosphorylation and that phorbol esters can influence the responsiveness of hormone-sensitive adenylate cyclase in certain cell types.
A human c-erbA oncogene homologue is closely proximal to the chromosome 17 breakpoint in acute promyelocytic leukemia.
Authors: Dayton A I , Selden J R , Laws G , Dorney D J , Finan J , Tripputi P , Emanuel B S , Rovera G , Nowell P C , Croce C M
Published: May 2006

A human cDNA library was screened for sequences homologous to the erbA gene of avian erythroblastosis virus (AEV). One such clone, cHerbA-1, was used to map the chromosomal location of highly homologous human sequences that were found to be present on chromosome 17 as judged by Southern blot screening of a panel of mouse-human hybrid cell lines segregating human chromosomes. cHerbA-1 was hybridized in situ to metaphase chromosomes from a normal male subject and from a female patient with an acute promyelocytic leukemia (APL) having the typical t(15;17) translocation. The results localized the cellular c-erbA sequences on chromosome 17 to the q21-q24 region of normal chromosomes and indicated that the c-erbA sequences remained on the 17q- chromosome in the APL cells, suggesting that they could be assigned to the 17(q21-q22) region. For additional data, we hybridized human neoplastic cells derived from a poorly differentiated acute leukemia carrying a t(17;21) translocation with thymidine kinase (TK)-deficient LMTK- mouse cells. A resulting hybrid, containing only the 21q+ chromosome, did not have human c-erbA sequences. Since the breakpoint on 17q in this translocation was similar to that in the APL t(15;17) translocation, this supported the assignment of c-erbA to the q21-q22 region of chromosome 17. The apparent close proximity of the c-erbA sequences to the chromosomal breakpoints in these two leukemias suggests a possible role for this oncogene homologue in the development of these neoplasms.
Aberrant expression of an amplified c-myb oncogene in two cell lines from a colon carcinoma.
Authors: Alitalo K , Winqvist R , Lin C C , de la Chapelle A , Schwab M , Bishop J M
Published: May 2006

Two cell lines (COLO 201 and COLO 205) derived independently from a single adenocarcinoma of the human colon each harbored an approximately 10-fold amplification of the cellular oncogene c-myb and a proportional abundance of the 4-kilobase mRNA derived from c-myb. By contrast, expression of c-myb could not be detected in cells from a variety of other solid tumors, including other colon carcinomas. Analysis of the amplified DNA with restriction endonucleases failed to reveal any topographical abnormalities within c-myb. Neither COLO 201 nor COLO 205 carry the double minute chromosomes and homogeneously staining regions of chromosomes that frequently serve as karyotypic signatures of amplified DNA. Instead, amplified c-myb is carried on what appear to be disomic or trisomic copies of the same anomalous marker chromosome that is characteristic of both COLO 201 and COLO 205. The karyological origin of this abnormal chromosome is not presently apparent. Our findings show c-myb expression by cells outside of the hemopoietic lineage, raise the possibility that amplification and/or ectopic expression of c-myb may have contributed to the genesis of the tumor from which the cells of COLO 201 and COLO 205 arose, and suggest that amplification of cellular oncogenes may be a more common factor in tumorigenesis than might have been suspected from available karyological data.
Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine: characterization and localization of receptor binding sites in rat and human brain.
Authors: Javitch J A , Uhl G R , Snyder S H
Published: May 2006

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces neuropathologic and clinical abnormalities in humans and animals that closely resemble idiopathic Parkinson disease. [3H]MPTP binds with high affinity (Kd = 28 X 10(-9) M) to brain membranes. The chemical specificity of the binding sites corresponds to structure-activity requirements for neurotoxicity. Autoradiographic studies in human brain show very high receptor densities in the caudate, substantia nigra, and locus coeruleus, which may explain the neurotoxic and neurochemical sequelae of MPTP administration. In contrast to the human, rat substantia nigra and caudate display only moderate receptor concentrations. This species difference may explain the difficulty in producing selective nigrostriatal degeneration in rats. Sites densely labeled in rat brain include the locus coeruleus, interpeduncular nucleus, arcuate and periventricular hypothalamic nuclei, and the subfornical organ.
Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).
Authors: Kasarda D D , Okita T W , Bernardin J E , Baecker P A , Nimmo C C , Lew E J , Dietler M D , Greene F C
Published: May 2006

The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently.
Crystallization of reaction center from Rhodopseudomonas sphaeroides: preliminary characterization.
Authors: Allen J P , Feher G
Published: May 2006

Reaction centers (RCs), integral membrane proteins that mediate the conversion of light into chemical energy, were crystallized by two different vapor diffusion techniques. In one method, small amphipathic molecules (1,2,3-heptanetriol and triethylammonium phosphate) were added to the RCs that had been solubilized in detergent. In the second method, crystallization occurred near the phase boundaries of a two-phase system created by the addition of polyethylene glycol and NaCl to RCs in octyl beta-D-glucoside. Several different crystal forms were obtained; two were analyzed by x-ray diffraction. One was monoclinic (space group P2) with beta = 105 degrees, and a = 70 A, b = 105 A, and c = 85 A, two RCs per unit cell, and one RC per asymmetric unit; the crystal diffracted to 3.5 A at 17 degrees C. The other crystal form was orthorhombic (space group C222) with a = 185 A, b = 170 A, and c = 105 A, with eight RCs per unit cell and one RC per asymmetric unit. Reversible light-induced EPR signals of the primary donor (bacteriochlorophyll dimer) showed that the RCs in the crystal were fully active. From the angular dependence of the EPR signal the molecular g anisotropy of the bacteriochlorophyll dimer was deduced to be g perpendicular - g parallel = (64 +/- 3) X 10(-5). Linear dichroism measurements were performed on the monoclinic crystal. The two bands at 535 and 544 nm assigned to the Qx transitions of the bacteriopheophytins were resolved and preliminary orientations of some of the pigments were obtained.