bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.
Authors: Jehuda-Cohen T , Slade B A , Powell J D , Villinger F , De B , Folks T M , McClure H M , Sell K W , Ahmed-Ansari A
Published: May 2006

Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals.
Alkylation interference identifies essential DNA contacts for sequence-specific binding of the eukaryotic transcription factor C/EBP.
Authors: Nye J A , Graves B J
Published: May 2006

Transcriptional regulators containing a "leucine zipper" and a flanking basic domain belong to a recently identified class of DNA-binding proteins. We have mapped the essential DNA contacts of one member of this group, the eukaryotic transcription factor C/EBP. Methylation and ethylation interference experiments detected major groove contacts over a full turn of the DNA helix on both an asymmetric and a symmetric C/EBP binding site. The contacts essential for C/EBP binding have two-fold symmetry yet differ significantly from the contacts of other dimeric DNA-binding proteins, including those bearing helix-turn-helix motifs and the type I restriction endonuclease EcoRI.
A cystic fibrosis pancreatic adenocarcinoma cell line.
Authors: Schoumacher R A , Ram J , Iannuzzi M C , Bradbury N A , Wallace R W , Hon C T , Kelly D R , Schmid S M , Gelder F B , Rado T A
Published: May 2006

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.
Role of instability in the cis action of the insertion sequence IS903 transposase.
Authors: Derbyshire K M , Kramer M , Grindley N D
Published: May 2006

An unusual subset of DNA-binding proteins, termed cis-acting proteins, has been shown to act preferentially at their site of synthesis; the transposases of several bacterial insertion sequences (ISs) fall into this class. The transposase of IS903 exhibits a strong preference for action in cis: complementation of defective transposons in trans occurs at less than 1%. Furthermore, transposition mediated by transposase acting in cis is extremely sensitive to the distance between the 3' end of the transposase gene and the nearest transposon inverted repeat; we find that an insertion of 1 kilobase of DNA reduces transposition to 1-2% of control levels. Here we show that there is a strong correlation between the stability of transposase and its ability to act in trans. We found that the wild-type transposase is a very unstable protein with a physical half-life of about 3 min. However, a transposase-beta-galactosidase fusion protein has a much greater half-life and can act equally well in cis or in trans. In addition, the native transposase is stabilized in lon- strains of Escherichia coli, and, in these protease-deficient strains, trans action of transposase is increased 10- to 100-fold. These results suggest that instability of the IS903 transposase is a major determinant of its cis action and that the La protease, product of the lon gene, is an important determinant of transposase instability.
Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro.
Authors: Falk R J , Terrell R S , Charles L A , Jennette J C
Published: May 2006

Anti-neutrophil cytoplasmic autoantibodies (ANCA) are in the circulation of most patients with pauci-immune necrotizing vasculitis and pauci-immune crescentic glomerulonephritis. The current study demonstrates an effect of these autoantibodies on neutrophil function in vitro. ANCA cause normal human neutrophils to undergo an oxidative burst and degranulate. Both ANCA phenotypes (i.e., cytoplasmic-pattern ANCA and myeloperoxidase-specific ANCA) induce neutrophil activation. ANCA sera and purified immunoglobulins significantly increase the release of reactive oxygen species when compared with controls. ANCA, in a dose-dependent manner, induce the release of primary granule contents. These effects are markedly enhanced by priming neutrophils with tumor necrosis factor. Flow cytometry studies demonstrate the presence of myeloperoxidase on the surface of neutrophils after cytokine priming, indicating that primed neutrophils have ANCA antigens at their surfaces to interact with ANCA. These observations suggest an in vivo pathogenetic role for ANCA. We propose that, in patients with necrotizing vasculitis, ANCA-induced release of toxic oxygen radicals and noxious granule enzymes from cytokine-primed neutrophils could be mediating vascular inflammation.
Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H).
Authors: McNeil H P , Simpson R J , Chesterman C N , Krilis S A
Published: May 2006

Anti-phospholipid (aPL) antibodies that exhibit binding in cardiolipin (CL) ELISA can be purified to greater than 95% purity by sequential phospholipid affinity and ion-exchange chromatography. However, these highly purified aPL antibodies do not bind to the CL antigen when assayed by a modified CL ELISA in which the blocking agent does not contain bovine serum, nor do they bind to phospholipid affinity columns. Binding to the phospholipid antigen will only occur if normal human plasma, human serum, or bovine serum is present, suggesting that the binding of aPL antibodies to CL requires the presence of a plasma/serum cofactor. Using sequential phospholipid affinity, gel-filtration, and ion-exchange chromatography, we have purified this cofactor to homogeneity and shown that the binding of aPL antibodies to CL requires the presence of this cofactor in a dose-dependent manner. N-terminal region sequence analysis of the molecule has identified the cofactor as beta 2-glycoprotein I (beta 2GPI) (apolipoprotein H), a plasma protein known to bind to anionic phospholipids. These findings indicate that the presence of beta 2GPI is an absolute requirement for antibody-phospholipid interaction, suggesting that bound beta 2GPI forms the antigen to which aPL antibodies are directed. Recent evidence indicates that beta 2GPI exerts multiple inhibitory effects on the coagulation pathway and platelet aggregation. Interference with the function of beta 2GPI by aPL antibodies could explain the thrombotic diathesis seen in association with these antibodies.
Differential protein expression by Shigella flexneri in intracellular and extracellular environments.
Authors: Headley V L , Payne S M
Published: May 2006

Shigellae were intrinsically radiolabeled with [35S]methionine either extracellularly or while multiplying within infected HeLa cell monolayers. A complex pattern of suppression and induction of proteins was observed. Proteins of approximately 97, 62, 58, 50, 25, and 18 kilodaltons (kDa) were induced in Shigella flexneri isolated from infected monolayers. Proteins of 100, 85, 70, 64, and 55 kDa were suppressed under the same conditions but were seen in cells labeled in the tissue culture medium alone. Protein expression during the stages of attachment, invasion, and intracellular multiplication was examined by pulse-labeling. The 58-kDa protein was induced only during invasion, and the 62- and 25-kDa proteins were induced only during intracellular multiplication. Shift into a minimal medium with ion concentrations and pH mimicking intracellular conditions and endosomal pH resulted in the induction of the 97- and 58-kDa proteins, and reduction of the intracellular-like medium with 2-mercaptoethanol resulted in the induction of the 97-, 50-, and 25-kDa proteins and suppression of the 55-kDa protein. Radioimmunoprecipitations of shigellae grown in vitro and in vivo revealed differential expression of immunogenic proteins. Proteins corresponding in size to IpaB (62 kDa), IpaC (42 kDa), and IpaD (38 kDa) were lost during intracellular multiplication, whereas another protein corresponding to IpaA (80 kDa) was found to increase under the same conditions.
Increased phosphorylation of elongation factor 2 during mitosis in transformed human amnion cells correlates with a decreased rate of protein synthesis.
Authors: Celis J E , Madsen P , Ryazanov A G
Published: May 2006

Elongation factor 2 was identified in the two-dimensional gel patterns of asynchronous human amnion cells (AMA) by comigration with purified rabbit reticulocyte elongation factor 2 and by two-dimensional gel immunoblot analysis using a specific rabbit polyclonal antibody. In all, four polypeptides were identified corresponding to isoelectric focusing polypeptides 2713 (95.0 kDa), 2714 (94.8 kDa), 3727 (94.8 kDa), and 3702 (93.6 kDa) (listed in order of decreasing pI values) in the computerized comprehensive two-dimensional gel data base of human AMA proteins. The relative proportion of two of these variants (isoelectric focusing polypeptides 3727 and 3702), which are phosphorylated, increased dramatically during mitosis. Since phosphorylation is known to render elongation factor 2 inactive in translation, this observation may partly explain the decline in the rate of protein synthesis observed during cell division.
Serum amine oxidase activity contributes to crisis in mouse embryo cell lines.
Authors: Parchment R E , Lewellyn A , Swartzendruber D , Pierce G B
Published: May 2006

This paper reports the results of experiments to test the hypothesis that crisis of spontaneous transformation is caused by the hydrogen peroxide and/or aldehydes generated from endogenous polyamines by serum amine oxidase [amine: oxygen oxidoreductase (deaminating), EC 1.4.3.6]. After 4-5 weeks of culture, crisis occurred in 16 of 29 cell lines derived from limb buds of embryos from SJL/J, C3H, and CD-1 mice. In contrast, after the same time in culture but in medium supplemented with aminoguanidine, which inhibits serum amine oxidase, crisis occurred in only 1 of 41 cell lines. Protection against crisis was maximal in cell lines of SJL/J embryos, in which the incidence of crisis fell from 7 of 9 in untreated controls of 0 to 12 in the presence of 2 mM aminoguanidine. 2-Mercaptoethanol at 150-300 microM, which protects cells from serum amine oxidase-dependent polyamine toxicity, also protected the cell lines against crisis. These protected cell lines retained proliferative potential, diploid DNA content, and the mixture of cell types found in the primary cultures. These results indicate that cytotoxic catabolites generated by serum amine oxidase caused at least a large portion, but perhaps not all, of the cellular damage that leads to crisis in mouse embryo cell lines.
Confirmation of dust condensation in the ejecta of supernova 1987a.
Authors: Gehrz R D , Ney E P
Published: May 2006

Shortly after its outburst, we suggested that supernova 1987a might condense a dust shell of substantial visual optical thickness as many classical novae do and predicted that dust might form within a year after the explosion. A critical examination of recent optical and infrared observations reported by others confirms that dust grains had begun to grow at a temperature of 1000 K after 300 days and that the dust shell had become optically thick by day 600. After day 600, the infrared luminosity closely followed the intrinsic luminosity expected for thermalized 56Co gamma rays, demonstrating that the luminosity is powered by radioactivity and that the dust is outside the radioactivity zone. The infrared luminosity sets an upper limit to the soft intrinsic bolometric luminosity of a pulsar central engine. This upper limit for the pulsar in supernova 1987a is the same luminosity as the Crab pulsar has today 936 years after its formation. It is unlikely that the rotation rate for a pulsar in supernova 1987a can be much higher than approximately 30 revolutions per sec. The relatively long time required for the shell to grow to maximum optical depth as compared with the dust in nova shells may be related to the relatively low outflow velocity of the condensible ejecta.
Potentiation of a polyadenylylation site by a downstream protein-DNA interaction.
Authors: Dorsett D
Published: May 2006

The gypsy retroposon of Drosophila melanogaster contains a sequence that potentiates upstream polyadenylylation sites. In contrast to other sequences that influence poly(A) site use, it appears to operate at the level of the DNA template. Nuclear extracts contained protein that bound to a repeated motif in the DNA. Flies with mutations that reduced transcripts polyadenylylated in the 5' long terminal repeat of gypsy contained less DNA-binding activity than wild type. A change in the repeat motif reduced both protein binding and poly(A) site potentiation. These findings provide evidence that DNA-binding proteins can regulate polyadenylylation sites.
Fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis revealed by organelle-specific antibodies.
Authors: Mourelatos Z , Adler H , Hirano A , Donnenfeld H , Gonatas J O , Gonatas N K
Published: May 2006

Many studies have established the central involvement of the Golgi apparatus in the transport and processing of plasma membrane, lysosomal, and secreted proteins. The Golgi apparatus of neurons is also involved in the axoplasmic flow of fast-moving macromolecules and in the orthograde, retrograde, and transsynaptic transport of exogenous ligands. Markers of the Golgi apparatus, based on traditional methods of enzyme cytochemistry, are not applicable to human tissues obtained at autopsy. For that reason, the Golgi apparatus of brain cells has not been examined adequately in diseases of the human nervous system. Here we report that an antiserum raised against MG-160, a 160-kDa sialoglycoprotein of medial cisternae of the Golgi apparatus of several rat cells, is a specific and easily reproducible immunocytochemical marker of the Golgi apparatus of human neurons and other cells obtained at autopsy. Application of this probe in amyotrophic lateral sclerosis has shown a fragmentation of the Golgi apparatus in motor neurons similar to that induced by depolymerization of microtubules. We suggest that the fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis has functional implications because significant reductions of secretion of insulin and immunoglobulins have been observed in islet cells and plasma cells, respectively, treated with microtubule-disrupting agents.
The microenvironment created by grafting rostral half-somites is mitogenic for neural crest cells.
Authors: Goldstein R S , Teillet M A , Kalcheim C
Published: May 2006

Chicken embryo neural crest cells that migrate into a paraxial mesoderm constructed of multiple rostral half-somites from quail embryos form unsegmented "polyganglia," instead of distinct dorsal root ganglia (DRG). We report here that the environment that is created by grafting rostral somitic (RS) moieties not only is permissive for neural crest cell migration and consequent DRG formation but also is mitogenic for the DRG precursor cells. On embryonic day 3.5 (E3.5), 1 day after surgery, there is a 42% average increase in volume of the polyganglia compared with the corresponding DRG on the unoperated side. The volume increase is accounted for by an increased number of DRG cells--an average of 46% more cells are found in the polyganglia. The increases in volume and cell number are still present a day later at E4.5 (38% and 52%, respectively) and are observed in both limb-forming and non-limb-forming regions of the embryonic axis. The mechanism for this increase in cell number and volume in the polyganglia is enhanced proliferative activity. On E3.5 the proportion of cells incorporating thymidine of the total DRG cell number is 45% higher in the polyganglia than the control side, when embryos are given a short pulse before sacrifice. This indicates that rostral sclerotomal environment stimulates the crest cells to proliferate. The difference in volume between the polyganglia and the normal DRG continues to grow until at least E8, when the polyganglia are twice as large as the control DRG. The continued increase in volume can also be accounted for by the mitogenic effect of the RS grafts, since on E4.5 the percentage of thymidine-labeled cells compared with the total cell number in DRG is 28% higher in the polyganglia than in control ganglia. This study demonstrates that the somitic microenvironment regulates the proliferation of neural crest cells in the nascent DRG.
rbcL sequence divergence and phylogenetic relationships in Saxifragaceae sensu lato.
Authors: Soltis D E , Soltis P S , Clegg M T , Durbin M
Published: May 2006

Phylogenetic relationships are often poorly understood at higher taxonomic levels (family and above) despite intensive morphological analysis. An excellent example is Saxifragaceae sensu lato, which represents one of the major phylogenetic problems in angiosperms at higher taxonomic levels. As originally defined, the family is a heterogeneous assemblage of herbaceous and woody taxa comprising 15 subfamilies. Although more recent classifications fundamentally modified this scheme, little agreement exists regarding the circumscription, taxonomic rank, or relationships of these subfamilies. The recurrent discrepancies in taxonomic treatments of the Saxifragaceae prompted an investigation of the power of chloroplast gene sequences to resolve phylogenetic relationships within this family and between the Saxifragaceae and other major plant lineages. Sequence data from the gene rbcL (ribulose-1,5-bisphosphate carboxylase, large subunit) reveal that (i) Saxifragaceae sensu lato is at least paraphyletic, and probably polyphyletic, (ii) the genera Parnassia and Brexia are only distantly related to other members of Saxifragaceae, and (iii) representatives of the Solanaceae (subclass Asteridae) appear more closely related to Saxifragaceae (subclass Rosidase) than traditionally maintained. These data illustrate the value of chloroplast gene sequence data in resolving genetic, and hence phylogenetic, relationships among members of the most taxonomically complex groups.
Low-affinity placenta-derived receptors for human granulocyte-macrophage colony-stimulating factor can deliver a proliferative signal to murine hemopoietic cells.
Authors: Metcalf D , Nicola N A , Gearing D P , Gough N M
Published: May 2006

Retrovirally mediated introduction of a cDNA encoding a placenta-derived low-affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) into murine FDC-P1 hemopoietic cells allowed these cells to proliferate when stimulated by human GM-CSF. The expressed human receptors on cloned lines were of low affinity (Kd = 4-6 nM), were internalized, and did not interact with endogenous GM-CSF receptors. Concentrations of human GM-CSF of 6.5-13 nM were required to stimulate 50% maximal colony formation versus a concentration of murine GM-CSF of 6 pM; this difference is comparable with the difference in relative affinities of the human and murine receptors for their respective ligands. If maintained in murine GM-CSF, cells able to bind or respond to human GM-CSF were rapidly lost due to transcriptional inactivation of the inserted cDNA. The observations indicate that low-affinity receptors for human GM-CSF can deliver a proliferative signal in appropriate cells and that the signaling mechanisms are not species-specific.
cDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides.
Authors: Richter K , Egger R , Negri L , Corsi R , Severini C , Kreil G
Published: May 2006

We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin.
Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate synthase in tomato.
Authors: Van der Straeten D , Van Wiemeersch L , Goodman H M , Van Montagu M
Published: May 2006

1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14), the key enzyme in ethylene biosynthesis, was purified 5000-fold from induced tomato pericarp. ACC synthase activity was unambiguously correlated with a 45-kDa protein by two independent methods. Peptide sequences were obtained both from the N terminus after electroblotting and from tryptic peptides separated by reversed-phase chromatography. Mixed oligonucleotide probes were used to screen a lambda gt11 library prepared from RNA of induced pericarp tissue. Putative ACC synthase clones were isolated with a frequency of 0.01%. One of these contained a 1.9-kilobase insert with a single open reading frame encoding a polypeptide of 55 kDa. A second, partial cDNA clone was found that differed from the first one in 18% of its bases. Genomic Southern blotting suggests possible tandem organization of the two genes in tomato. The entire coding region was expressed in Escherichia coli and the denatured recombinant polypeptide was used to raise polyclonal antibodies. The antibody preparation both immunoinhibits and immunoprecipitates ACC synthase activity from an enriched tomato extract, confirming the identity of the clone. Northern blot analysis demonstrates that the ACC synthase messenger accumulation is coordinated with fruit ripening.
Use of second-site suppressor mutations in Drosophila to identify components of the transcriptional machinery.
Authors: Mortin M A
Published: May 2006

Isolation of second-site suppressor mutations provides a powerful method for identifying (i) genes that encode proteins that interact and (ii) domains within the interacting proteins that contact each other. Flies conditionally lethal because they carry mutations in the largest subunit of RNA polymerase II were mutagenized; ten million progeny were then screened for compensatory mutations. Eight intragenic and 10 extragenic suppressor mutations were recovered. Both the conditional lethality and premature termination of transcription caused by one mutation in the largest subunit of RNA polymerase II are compensated by an allele-specific suppressor mutation in the second-largest subunit of the enzyme.
Ubiquinol-10 is an effective lipid-soluble antioxidant at physiological concentrations.
Authors: Frei B , Kim M C , Ames B N
Published: May 2006

It is well known that ubiquinone-10 (coenzyme Q10, ubiquinone 50) acts as an electron carrier of the respiratory chain in mitochondria. In this paper we show that ubiquinol-10, the reduced form of ubiquinone-10, also efficiently scavenges free radicals generated chemically within liposomal membranes. Ubiquinol-10 is about as effective in preventing peroxidative damage to lipids as alpha-tocopherol, which is considered the best lipid-soluble antioxidant in humans. The number of radicals scavenged by each molecule of ubiquinol-10 is 1.1 under our experimental conditions. In contrast to alpha-tocopherol, ubiquinol-10 is not recycled by ascorbate. However, it is known that ubiquinol-10 can be recycled by electron transport carriers present in various biomembranes and possibly by some enzymes. We also show that ubiquinol-10 spares alpha-tocopherol when both antioxidants are present in the same liposomal membranes and that ubiquinol-10, like alpha-tocopherol, does not interact with reduced glutathione. Our data together with previous work on the antioxidant function of ubiquinol reported in the literature strongly suggest that ubiquinol-10 is an important physiological lipid-soluble antioxidant.
Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine.
Authors: Roederer M , Staal F J , Raju P A , Ela S W , Herzenberg L A , Herzenberg L A
Published: May 2006

We show that the stimulation of human immunodeficiency virus (HIV) brought about by tumor necrosis factor alpha and phorbol 12-myristate 13-acetate can be inhibited by adding N-acetyl-L-cysteine (NAC). NAC, which replenishes intracellular glutathione, effectively inhibits the tumor necrosis factor alpha- or phorbol ester-stimulated replication of HIV in acutely infected cell cultures. NAC also inhibits the cytokine-enhanced HIV long terminal repeat-directed expression of beta-galactosidase in in vitro HIV model systems. These results show that intracellular thiol levels influence HIV production. Furthermore, because NAC reverses tumor necrosis factor alpha toxicity both in cells and in animals and is a well-known drug that can be administered orally without known toxicity in humans, these results suggest that NAC is a possible therapeutic agent in AIDS.
Frameshift errors initiated by nucleotide misincorporation.
Authors: Bebenek K , Kunkel T A
Published: May 2006

Studies presented here on the fidelity of DNA synthesis in vitro support the hypothesis that a classical base-substitution intermediate (i.e., a misincorporated nucleotide) can yield a frameshift mutation. By using a fidelity assay specifically designed to detect minus-one-base errors, nucleotide substrate pool imbalances that have previously been shown to increase the rate of misincorporation are now shown to also increase minus-one-base frameshift error rates. Examination of the specificity of the errors produced in reactions with various dNTP pool imbalances and various DNA templates revealed that template nucleotides were preferentially lost when they had as a 5' neighbor a nucleotide complementary to the dNTP provided in excess. This suggests that when a misincorporated nucleotide is complementary to the next nucleotide in the template, a misaligned intermediate containing a correct terminal base pair can form and be extended by a DNA polymerase, leading to a frameshift mutation. We present evidence that the proposed mechanism may operate in vivo and discuss the implications of this model for frameshift mutations induced by DNA damage.
Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences.
Authors: Webb B A , Summers M D
Published: May 2006

Endoparasitic wasps of lepidopteran insects must induce changes in host immunity and development to survive. Depending on the species, this may require wasp venom proteins and/or a polydnavirus. We describe an immunological and genetic relationship between the Campoletis sonorensis polydnavirus and the wasp's venom gland. Monoclonal antibodies raised against venom glands recognized epitopes conserved on several polydnavirus proteins and on multiple wasp oviduct and venom proteins. The viral envelope proteins had molecular masses of 16, 20, 45, and 50 kDa, while a complex of at least five immunoreactive venom-gland and soluble oviduct proteins ranged in size from 24 to 36 kDa. Since the conserved epitopes were present on the viral envelope, neutralization assays were performed. Monoclonal antibodies added to purified virus blocked the normal viral inhibition of host growth and development. To determine whether venom mRNA and viral genes were also related, venom-related cDNA clones were isolated from the wasp oviduct with a venom-gland cDNA probe. Venom-related viral clones were then identified and selected from a viral genomic library and from a parasitized Heliothis virescens cDNA library. Venom-related mRNAs were expressed in the venom gland, the oviduct, and the parasitized host. We propose that the immunological relationship between venom and viral proteins, and the hybridization of venom and viral genes, may reflect an evolutionary relationship in which venom gene homologs were incorporated into the viral genome, thereby allowing viral expression of venom-related genes and enhancing parasite survival.
Amplification and detection of lentiviral DNA inside cells.
Authors: Haase A T , Retzel E F , Staskus K A
Published: May 2006

Visna virus and human immunodeficiency virus are prototypes of animal and human lentiviruses, respectively, that persist and are disseminated despite the host immune response because cells in the tissues and the bloodstream harbor viral genomes in a covert state. To facilitate identification of these latently infected cells, the polymerase chain reaction has been adapted to amplify viral DNA in fixed cells for detection by in situ hybridization. By using a multiple primer set that generates DNA segments with overlapping cohesive termini, visna virus DNA can be amplified, retained, and detected in infected cells with sensitivities that exceed those of existing methods by more than 2 orders of magnitude. This advance in single-cell technology should prove useful in diagnosing and gaining insight into the pathogenesis of viral infections and provide new opportunities to look for viruses in chronic diseases of unknown etiology.
Stable expression of functional mitochondrial uncoupling protein in Chinese hamster ovary cells.
Authors: Casteilla L , Blondel O , Klaus S , Raimbault S , Diolez P , Moreau F , Bouillaud F , Ricquier D
Published: May 2006

The mitochondrial uncoupling protein (UCP) is a membranous proton carrier exclusively synthesized in brown adipocytes. The cDNA for the rat UCP was placed in an expression vector and transfected into mammalian cells. Its expression was tested in transiently transfected CHO cells. In these cells the UCP was detected in mitochondria by using antibodies. Permanent expression of the UCP was achieved in stable transformed CHO cell lines. In these cells the UCP was characterized in mitochondrial membranes, by using antibodies and hydroxyapatite purification. The protein expressed in CHO cells displayed the functional characteristics of brown adipocyte UCP. It induced the uncoupling of respiration in isolated CHO mitochondria. The membrane potential of transformed mitochondria was also significantly lowered, as a result of the proton translocating activity of the UCP. GDP is known to inhibit the proton pathway in brown fat mitochondria. Addition of GDP to CHO mitochondria containing UCP resulted in a recoupling of respiration and an increase in membrane potential. Thus we conclude that functional UCP is expressed in CHO cells and that the insertion of the UCP alone in any mitochondria is sufficient to induce the uncoupling of respiration. This approach should allow studies on the structure-function relationship of the UCP and of several other related mitochondrial carriers.
Accelerator mass spectrometry in biomedical dosimetry: relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA.
Authors: Turteltaub K W , Felton J S , Gledhill B L , Vogel J S , Southon J R , Caffee M W , Finkel R C , Nelson D E , Proctor I D , Davis J C
Published: May 2006

Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a 14C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations (10Be, 14C, 26Al, 41Ca, and 129I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of the technology. In this study, the relationship between exposure to low levels of 2-amino-3,8-dimethyl[2-14C]imidazo[4,5-f]quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Instrument reproducibility in this study is 2%; sensitivity is 1 adduct per 10(11) nucleotides. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, we demonstrate at least 1 order of magnitude improvement over the best adduct detection sensitivity reported to date and 3-5 orders of magnitude improvement over other methods used for adduct measurement. An additional improvement of 2 orders of magnitude in sensitivity is suggested by preliminary experiments to develop bacterial hosts depleted in radiocarbon. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.
Origin of variability in quantal size in cultured hippocampal neurons and hippocampal slices.
Authors: Bekkers J M , Richerson G B , Stevens C F
Published: May 2006

The size of synaptic quanta has been found to display considerable variation in cultured hippocampal neurons, but the source of this variability was previously unknown. We have now compared the properties of locally evoked miniature excitatory postsynaptic currents in cultured hippocampal neurons and in thin hippocampal slices using whole-cell patch-clamp recordings. The variability in miniature excitatory postsynaptic current size was similar in both preparations and occurred in cultured neurons when only one or a few synaptic boutons were stimulated. Thus, the variability in miniature excitatory postsynaptic current amplitude is not an artifact of cultured neurons and arises predominantly from variability within a single bouton. Possible origins of this variability are discussed.
Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents.
Authors: Dolan M E , Moschel R C , Pegg A E
Published: May 2006

O6-Alkylguanine-DNA alkyltransferase was rapidly and irreversibly inactivated by exposure to O6-benzylguanine or the p-chlorobenzyl and p-methylbenzyl analogues. This inactivation was much more rapid than with O6-methylguanine: incubation with 2.5 microM O6-benzylguanine led to more than a 90% loss of activity within 10 min, whereas 0.2 mM O6-methylguanine for 60 min was required for the same reduction. O6-Benzylguanine was highly effective in depleting the alkyltransferase activity of cultured human colon tumor (HT29) cells. Complete loss of activity was produced within 15 min after addition of O6-benzylguanine to the culture medium and a maximal effect was obtained with 5 microM. In contrast, at least 100 microM O6-methylguanine for 4 hr was needed to get a maximal effect, and this reduced the alkyltransferase by only 80%. Pretreatment of HT29 cells with 10 microM O6-benzylguanine for 2 hr led to a dramatic increase in the cytotoxicity produced by the chemotherapeutic agents 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) or 2-chloroethyl(methysulfonyl)methanesulfonate (Clomesone). Administration of O6-benzylguanine to mice at a dose of 10 mg/kg reduced alkyltransferase levels by more than 95% in both liver and kidney. These results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be valuable to increase the chemotherapeutic effectiveness of chloroethylating agents.
Tumor necrosis factor alpha modifies agonist-dependent responses in human neutrophils by inducing the synthesis and myristoylation of a specific protein kinase C substrate.
Authors: Thelen M , Rosen A , Nairn A C , Aderem A
Published: May 2006

Tumor necrosis factor alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS) induce the synthesis and cotranslational myristoylation of an 82-kDa specific protein kinase C substrate in human neutrophils. The myristic acid is covalently bound via a hydroxylamine-resistant amide linkage to the N-terminal glycine of the protein. The isoelectric point of the protein is at pH 4.6. The protein is rapidly phosphorylated when neutrophils are stimulated with chemotactic agonists or with phorbol 12-myristate 13-acetate, an activator of protein kinase C, and displays two characteristic phosphopeptides in one- and two-dimensional separation systems. Identical phosphopeptides were detected when the 82-kDa protein was phosphorylated in vitro with purified kinase C. The 82-kDa protein was immunoprecipitated by a polyclonal antiserum raised against the 87-kDa specific protein kinase C substrate from bovine brain. From these biochemical and immunological criteria it is concluded that the 82-kDa protein is the human neutrophil homolog of MARCKS, the myristoylated, alanine-rich C kinase substrate previously described in bovine and rat brain and in murine fibroblasts and macrophages. TNF-alpha and LPS prime human neutrophils for potentiated protein kinase C-dependent responses such as the respiratory burst and exocytosis. Consistent with this, these mediators do not induce the phosphorylation of MARCKS but prime the neutrophils for enhanced phosphorylation of this protein when the cells subsequently encounter activators of protein kinase C. This increase in MARCKS phosphorylation can be explained by the elevated levels of the protein observed in TNF-alpha- or LPS-treated neutrophils. Indeed, MARCKS constitutes 90% of all proteins synthesized in response to TNF-alpha or LPS. These data strongly suggest that MARCKS acts as a critical effector molecule in the transduction pathway of these important inflammatory mediators.
Intrinsic B-cell hyporesponsiveness accounts for self-tolerance in lysozyme/anti-lysozyme double-transgenic mice.
Authors: Adams E , Basten A , Goodnow C C
Published: May 2006

In double-transgenic mice expressing a gene construct encoding hen egg lysozyme as well as rearranged anti-lysozyme antibody genes, large numbers of anti-lysozyme B cells are present in peripheral lymphoid tissues but are profoundly tolerant. The cellular basis for this form of non-deletional self-tolerance was explored. The tolerant anti-lysozyme B cells from double-transgenic mice were found to produce much less antibody than nontransgenic controls in T-cell-dependent antigen-specific responses, in adoptive transfer in vivo, and in hanging-drop cultures in vitro, as well as in response to stimulation with the nonspecific mitogen lipopolysaccharide. The diminished responsiveness of the tolerant B cells was not due to a reduction in the number of responding B-cell precursors per se nor were suppressor cells detected in titration, depletion, or mixing experiments. Nondeletional tolerance in this model, therefore, appears to result from an intrinsic functional change in the self-reactive B cells themselves.
Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.
Authors: Gietl C
Published: May 2006

The isolation and sequence of a cDNA clone encoding the complete glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] of watermelon cotyledons are presented. Partial cDNA clones were synthesized in a three part strategy, taking advantage of the polymerase chain reaction technology with oligonucleotides based on directly determined amino acid sequences. Subsequently, the complete clone for gMDH was synthesized with a sense primer corresponding to the nucleotide sequence of the N-terminal end of pre-gMDH and an antisense primer corresponding to the adenylylation site found in the mRNA. The amino acids for substrate and cofactor binding identified by x-ray crystallography for pig heart cytoplasmic malate dehydrogenase are conserved in the 319-amino-acid-long mature plant enzyme. The pre-gMDH contains an N-terminal transit peptide of 37 residues. It has a net positive charge, lacks a long stretch of hydrophobic residues, and contains besides acidic amino acids a cluster of serine residues. This N-terminal extension is cleaved off upon association with or import into glyoxysomes. It contains a putative AHL topogenic signal for microbody import and has no sequence similarity to the 27-residue-long presequence of the watermelon mitochondrial malate dehydrogenase precursor.
A distinctive family of embryonic protein-tyrosine kinase receptors.
Authors: Pasquale E B
Published: May 2006

Two closely related protein-tyrosine kinases with the characteristics of growth factor receptors were identified by screening a chicken embryo cDNA expression library with anti-phosphotyrosine antibodies and were designated Cek2 and Cek3 (chicken embryo kinases 2 and 3). Cek2 and Cek3 are structurally related to Cek1, a chicken basic fibroblast growth factor receptor, and presumably represent receptors for basic fibroblast growth factor-related molecules. The identification of Cek2 and Cek3 establishes the existence of a family of protein-tyrosine kinases that includes Cek1 and that is likely to be implicated in the control of developmental processes. Among protein-tyrosine kinases, this family of receptors, which may include other as yet unknown members, is most closely related to the protooncogene product Ret and the platelet-derived growth factor receptor family.
Mismatch-specific thymine DNA glycosylase and DNA polymerase beta mediate the correction of G.T mispairs in nuclear extracts from human cells.
Authors: Wiebauer K , Jiricny J
Published: May 2006

To avoid the mutagenic effect of spontaneous hydrolytic deamination of 5-methylcytosine, G.T mispairs, arising in DNA as a result of this process, should always be corrected to G.C pairs. We describe here the identification of a DNA glycosylase activity present in nuclear extracts from HeLa cells, which removes the mispaired thymine to generate an apyrimidinic (AP) site opposite the guanine. We further show, using a specific antibody and inhibitors, that the single nucleotide gap, created upon processing of the AP site, is filled in by DNA polymerase beta. This finding substantiates the proposed role of this enzyme in short-patch DNA repair.
Generation of collagenase-resistant collagen by site-directed mutagenesis of murine pro alpha 1(I) collagen gene.
Authors: Wu H , Byrne M H , Stacey A , Goldring M B , Birkhead J R , Jaenisch R , Krane S M
Published: May 2006

Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, P1-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine pro alpha 1(I) collagen (Col1a1) gene. These mutant constructs have been transfected into homozygous Mov13 fibroblasts that do not express the endogenous Col1a1 gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Ile (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for Ile (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for Ile at position 776. The wild-type alpha 2(I) chains derived from the endogenous Col1a2 gene were also resistant to enzyme digestion when they were complexed with the mutant alpha 1(I) chains, indicating that the presence of normal alpha 1(I) sequences is critical for cleavage of the alpha 2(I) chains in the type I heterotrimer.
Down-regulation of voltage-dependent sodium channels initiated by sodium influx in developing neurons.
Authors: Dargent B , Couraud F
Published: May 2006

To address the issue of whether regulatory feedback exists between the electrical activity of a neuron and ion-channel density, we investigated the effect of Na(+)-channel activators (scorpion alpha toxin, batrachotoxin, and veratridine) on the density of Na+ channels in fetal rat brain neurons in vitro. A partial but rapid (t1/2, 15 min) disappearance of surface Na+ channels was observed as measured by a decrease in the specific binding of [3H]saxitoxin and 125I-labeled scorpion beta toxin and a decrease in specific 22Na+ uptake. Moreover, the increase in the number of Na+ channels that normally occurs during neuronal maturation in vitro was inhibited by chronic channel activator treatment. The induced disappearance of Na+ channels was abolished by tetrodotoxin, was found to be dependent on the external Na+ concentration, and was prevented when either choline (a nonpermeant ion) or Li+ (a permeant ion) was substituted for Na+. Amphotericin B, a Na+ ionophore, and monensin were able to mimick the effect of Na(+)-channel activators, while a KCl depolarization failed to do this. This feedback regulation seems to be a neuronal property since Na(+)-channel density in cultured astrocytes was not affected by channel activator treatment or by amphotericin B. The present evidence suggests that an increase in intracellular Na+ concentration, whether elicited by Na(+)-channel activators or mediated by a Na+ ionophore, can induce a decrease in surface Na+ channels and therefore is involved in down-regulation of Na(+)-channel density in fetal rat brain neurons in vitro.