bluuebunny/crossref_metadata_2025 huggingface dataset:
Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources.
Authors: Nelson D L , Ledbetter S A , Corbo L , Victoria M F , Ramírez-Solis R , Webster T D , Ledbetter D H , Caskey C T
Published: May 2006

Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions.
Surface immunoglobulin-mediated B-cell activation in the absence of detectable elevations in intracellular ionized calcium: a model for T-cell-independent B-cell activation.
Authors: Brunswick M , June C H , Finkelman F D , Dintzis H M , Inman J K , Mond J J
Published: May 2006

We recently showed that anti-immunoglobulin conjugated to high molecular weight dextran is 1000-fold more mitogenic for B cells than unconjugated anti-immunoglobulin. This system serves as a model for T-cell-independent type 2 antigens such as haptenated Ficoll, dextran, and bacterial polysaccharides, which can also stimulate B-cell proliferation and antibody production at low concentrations. We show here that conjugated anti-immunoglobulin, at concentrations that stimulate significant increases in expression of major histocompatibility complex class II molecules and incorporation of thymidine into DNA, does not induce detectable modulation of surface immunoglobulin. These results indicate that the facilitated T-cell-independent B-cell activation by polysaccharide antigens may result from inability to modulate surface immunoglobulin, possibly resulting in persistent and/or repetitive signaling. Early large increases in Ca2+ and breakdown of inositol phospholipids presently thought to be involved in transduction of the mitogenic signal are not detectable at low concentrations of conjugated anti-immunoglobulin, raising the possibility that these biochemical events may not in fact be central to this signaling pathway.
Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein.
Authors: Javaherian K , Langlois A J , McDanal C , Ross K L , Eckler L I , Jellis C L , Profy A T , Rusche J R , Bolognesi D P , Putney S D
Published: May 2006

The principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) is located in the external envelope protein, gp120, and has previously been mapped to a 24-amino acid-long sequence (denoted RP135). We show here that deletion of this sequence renders the envelope unable to elicit neutralizing antibodies. In addition, using synthetic peptide fragments of RP135, we have mapped the neutralizing determinant to 8 amino acids and found that a peptide of this size elicits neutralizing antibodies. This sequence contains a central Gly-Pro-Gly that is generally conserved between different HIV-1 isolates and is flanked by amino acids that differ from isolate to isolate. Antibodies elicited by peptides from one isolate do not neutralize two different isolates, and a hybrid peptide, consisting of amino acid sequences from two isolates, elicits neutralizing antibodies to both isolates. By using a mixture of peptides of this domain or a mixture of such hybrid peptides the type-specificity of the neutralizing antibody response to this determinant can perhaps be overcome.
Transient low-threshold Ca2+ current triggers burst firing through an afterdepolarizing potential in an adult mammalian neuron.
Authors: White G , Lovinger D M , Weight F F
Published: May 2006

In a variety of mammalian neurons, a brief depolarization generates an afterdepolarizing potential that triggers the firing of a short series or burst of action potentials. Although such burst firing is thought to contribute to the processing of neural information, the ionic currents that underlie this phenomenon have not been established. In whole-cell patch-clamp experiments on dorsal root ganglion neurons, we have found that the current that underlies this type of burst firing is a transient low-threshold (T-type) Ca2+ current. The data suggest that the T-type Ca2+ current may play an important role in the processing of information in the nervous system by virtue of its ability to elicit burst firing in neurons.
mRNAs for plasma membrane calcium pump isoforms differing in their regulatory domain are generated by alternative splicing that involves two internal donor sites in a single exon.
Authors: Strehler E E , Strehler-Page M A , Vogel G , Carafoli E
Published: May 2006

cDNA clones coding for human plasma membrane Ca2+ pump isoforms have been isolated from a fetal skeletal muscle cDNA library. Compared with the sequence of a teratoma cDNA-encoded pump these clones specify isoforms that contain either 29- or 38-amino acid insertions within the calmodulin-binding region. Replacement of two basic arginine residues by an aspartic acid and a glutamine residue could influence the binding of calmodulin to these isoforms. RNase mapping shows that RNA species containing the 29-residue-encoding insertion are particularly abundant in skeletal muscles. The sequences coding for the insertions are present on a single 154-base-pair exon, as demonstrated by an analysis of the corresponding genomic region, and they are included in their respective mRNAs by alternative splicing involving the differential usage of two internal "cryptic" donor splice sites in the presence of a nearby canonical one. Inclusion of the complete 154-base-pair exon results in an mRNA coding for a pump protein with a shorter C-terminal amino acid sequence that lacks a consensus site for phosphorylation by the cAMP-dependent kinase. Exclusion, inclusion, or partial inclusion of the same exon can thus lead to the production of four different mRNAs from a single gene. When expressed as protein, these mRNAs encode Ca2+ pump isoforms that differ in their C-terminal regulatory domains.
Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA.
Authors: Powell P A , Stark D M , Sanders P R , Beachy R N
Published: May 2006

Transgenic tobacco plants that express RNA sequences complementary to the tobacco mosaic virus (TMV) coat protein (CP) coding sequence with or without the tRNA-like structure at the 3' end of the TMV RNA were produced. Progeny of self-pollinated plants were challenged with TMV to determine their resistance to infection. Plants that expressed RNA sequences complementary to the CP coding region and the 3' untranslated region, including the tRNA-like sequences, were protected from infection by TMV at low levels of inoculum. However, plants that expressed RNA complementary to the CP coding sequence alone were not protected from infection. These results indicate that sequences complementary to the terminal 117 nucleotides of TMV, which include a putative replicase binding site, are responsible for the protection. However, the level of protection in these plants was considerably less than in transgenic plants that expressed the TMV CP gene and accumulated CP. Since the mechanisms of protection in the two systems are different, it may be possible to increase protection by introducing both sequences into transgenic plants.
Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region.
Authors: Ludwig S R , Habera L F , Dellaporta S L , Wessler S R
Published: May 2006

Previous studies have suggested that the R locus of maize is responsible for determining the temporal and spatial pattern of anthocyanin pigmentation in the plant. In this report we demonstrate that three members of the R gene family, P, S, and Lc, encode homologous transcripts 2.5 kilobases in length. The structure of one R gene, Lc, was determined by sequencing cDNA and genomic clones. The putative Lc protein, deduced from the cDNA sequence, is composed of 610 amino acids and has homology to the helix-loop-helix DNA-binding/dimerization motif found in the L-myc gene product and other regulatory proteins. It also contains a large acidic domain that may be involved in transcriptional activation. Consistent with its proposed role as a transcriptional activator is our finding that a functional R gene is required for the accumulation of transcripts of at least two genes in the anthocyanin biosynthetic pathway. We discuss the possibility that the diverse patterns of anthocyanin pigmentation conditioned by different R genes reflect differences in the R gene promoters rather than their gene products.
Phosphorothioate and cordycepin analogues of 2',5'-oligoadenylate: inhibition of human immunodeficiency virus type 1 reverse transcriptase and infection in vitro.
Authors: Montefiori D C , Sobol R W , Li S W , Reichenbach N L , Suhadolnik R J , Charubala R , Pfleiderer W , Modliszewski A , Robinson W E , Mitchell W M
Published: May 2006

Natural antiviral activity can be mediated by the interferon-induced synthesis of 2',5'-oligoadenylates (2-5As) and subsequent RNase L activation by these molecules. Analogues of 2-5A that are biologically active and metabolically stable were synthesized and analyzed for antiviral activity against the human immunodeficiency virus type 1 (HIV-1). Replacement of the 3' hydroxyl group of the adenosine moieties of 2-5A with hydrogen atoms (i.e., cordycepin analogues of 2-5A) converted authentic 2-5A trimer into anti-HIV-1 agents in vitro. These cordycepin analogues of 2-5A also inhibited partially purified HIV-1 reverse transcriptase. Introduction of chirality into the 2',5'-phosphodiester internucleotide linkages or 5'-phosphate moieties of the 2-5A molecule (i.e., phosphorothioate analogues of 2-5A) converted authentic 2-5A into more potent inhibitors of HIV-1 reverse transcriptase. However, these phosphorothioate 2-5As demonstrated little or no anti-HIV-1 activity in vitro. Thus, some analogues of 2-5A may form a class of anti-HIV-1 drugs with possible pleiotropic activities that include activation of latent RNase L and inhibition of reverse transcription.
Loss of heterozygosity on chromosome 1q in human breast cancer.
Authors: Chen L C , Dollbaum C , Smith H S
Published: May 2006

Cytogenetic markers involving the long arm of chromosome 1 are the most frequently observed karyotypic changes seen in breast cancer. Based on cytogenetic data, we have used polymorphic DNA markers to search for allelic losses at this chromosome region among 48 breast carcinomas. For SPTA1, allelic losses were seen in 6 of 26 (23%) informative carcinomas, while 3 of 13 (23%) and 5 of 19 (26%) informative patients showed losses at AT3 and D1S53, respectively. The background frequency of allelic loss was obtained from data using 3 other loci on the 1q arm and 2 on the p arm of chromosome 1. With these markers, only 6 of 62 informative patients (8%) showed an allelic loss, with the range being 0-13%. The allelic losses seen on 1q, which were found in 9 carcinomas, comprised an overlapping set; the common region deleted was approximately 26 centimorgans on the q arm of chromosome 1 (bands q23-32 between AT3 and D1S53). These results suggest that inactivation of a gene(s) located on 1q23-32 might contribute to the genesis of breast cancer.
Endothelin: visualization of mRNAs by in situ hybridization provides evidence for local action.
Authors: MacCumber M W , Ross C A , Glaser B M , Snyder S H
Published: May 2006

Endothelin (ET) is a recently identified vasoactive peptide with three isoforms for which three genes have been cloned. The cellular sites of synthesis of this peptide have not yet been identified in vivo. Using Northern blot analysis, we have detected two forms of ET mRNA in rat tissues: a 3.7-kilobase form in the kidney, eye, and brain, a 2.5-kilobase form in the intestine, and both forms in the lung. We have localized these forms of ET mRNA in several rat tissues using in situ hybridization. In the 19-day rat fetus, ET mRNA is highest in the lung, intestine, and meninges. At high resolution, ET mRNA is localized in the lung to respiratory epithelial cells of bronchioles and apparently in blood vessels. In adult tissues, ET mRNA is present throughout the lung, in the renal medulla vasa recta, and in the iris of the eye. ET mRNA is synthesized in close proximity to ET binding sites in many organs (e.g., lung, kidney, intestine, and eye), suggesting a local action of this peptide. However, in other areas (e.g., heart and renal cortex), ET binding sites are present in the absence of ET mRNA, suggesting an action of ET from the bloodstream or from neurons. Northern blot analysis of ET mRNA in microvascular endothelial cells in culture indicates that ET is synthesized in small blood vessels and regulated similarly to its regulation in large vessels. Our results provide evidence that ET, like other regulatory peptides, may serve in several tissues as a neuromodulator or local hormone.
gamma-Aminobutyric acid uptake by a bacterial system with neurotransmitter binding characteristics.
Authors: Guthrie G D , Nicholson-Guthrie C S
Published: May 2006

gamma-Aminobutyric acid (GABA), an amino acid, has been found in every class of living organisms. In higher organisms, GABA is a neurotransmitter and binds with high affinity and specificity to GABA receptors on neurons in a sodium-independent reaction that is saturable. The role of GABA in organisms lacking nervous tissue is not known. This report describes, in a strain of Pseudomonas fluorescens, a GABA uptake system with binding characteristics like those of the GABA (type A) brain receptor. The binding was saturable and specific for GABA, was sodium-independent, was of high affinity (Km = 65 nM), and was inhibited competitively by muscimol, a potent GABA analogue. The bacterial GABA system included a homogeneous binding site, and no cooperative interaction was found between sites. To our knowledge, such a system for GABA, or other neurotransmitters, in a bacterium has not been reported.
RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron.
Authors: Doudna J A , Cormack B P , Szostak J W
Published: May 2006

The group I self-splicing introns act at exon-intron junctions without recognizing a particular sequence. In order to understand splice-site selection, we have developed an assay system based on the Tetrahymena ribozyme to allow the study of numerous 5'-splice-site variants. Cleavage at the correct site requires formation of the correct secondary structure and occurs most efficiently within a 3-base-pair window centered on base pair 5 from the bottom of the P1 stem. Within this window the ribozyme recognizes and cleaves at a "wobble" base pair; the base pair above the cleavage site also influences splicing efficiency. The recognition of RNA structure rather than sequence explains the ability of these transposable introns to splice out of a variety of sequence contexts.
Artificial nucleosome positioning sequences.
Authors: Shrader T E , Crothers D M
Published: May 2006

We have used the emerging rules for the sequence dependence of DNA bendability to design and test a series of DNA molecules that incorporate strongly into nucleosomes. Competitive reconstitution experiments showed the superiority in histone octamer binding of DNA molecules in which segments consisting exclusively of A and T or G and C, separated by 2 base pairs (bp), are repeated with a 10-bp period. These repeated (A/T)3NN(G/C)3NN motifs are superior in nucleosome formation to natural positioning sequences and to other repeated motifs such as AANNNTTNNN and GGNNNCCNNN. Studies of different lengths of repetitive anisotropically flexible DNA showed that a segment of approximately 40 bp embedded in a 160-bp fragment is sufficient to generate nucleosome binding equivalent to that of natural nucleosome positioning sequences from 5S RNA genes. Bending requirements along the surface of the nucleosome seem to be quite constant, with no large jumps in binding free energy attributable to protein-induced kinks. The most favorable sequences incorporate into nucleosomes more strongly by 100-fold than bulk nucleosomal DNA, but differential bending free energies are small when normalized to the number of bends: a free energy difference of only about 100 cal/mol per bend (1 cal = 4.184 J) distinguishes the best bending sequences and bulk DNA. We infer that the distortion energy of DNA bending in the nucleosome is only weakly dependent on DNA sequence.
Loss of the MYC gene amplified in human HL-60 cells after treatment with inhibitors of poly(ADP-ribose) polymerase or with dimethyl sulfoxide.
Authors: Shima H , Nakayasu M , Aonuma S , Sugimura T , Nagao M
Published: May 2006

In HL-60 cells, a human promyelocytic leukemia cell line, the human c-myc gene, designated MYC, is amplified about 16-fold. On differentiation of the HL-60 cells into granulocytes induced by several inhibitors of poly(ADP-ribose) polymerase [NAD+ poly(adenosine diphosphate D-ribose)ADP-D-ribosyltransferase, EC 2.4.2.30] including benzamide, nicotinamide, coumarin, and 4-hydroxyquinazoline or dimethyl sulfoxide, some MYC loss was observed. In contrast, benzoic acid, a noninhibitory analogue of benzamide, did not induce either granulocytic differentiation or loss of MYC. Loss of MYC seems to be associated with granulocytic differentiation because the time course of its loss was similar to that of appearance of nitroblue tetrazolium-positive cells, mature granulocytes, and its loss was not observed on differentiation of HL-60 cells into macrophages induced by phorbol 12-myristate 13-acetate or teleocidin. The loss of MYC is not the reason for the down regulation of MYC expression observed within 1 hr after addition of inducers, since the loss of MYC was not detected by 1-day treatment with inducers.
Immature human thymocytes can be driven to differentiate into nonlymphoid lineages by cytokines from thymic epithelial cells.
Authors: Kurtzberg J , Denning S M , Nycum L M , Singer K H , Haynes B F
Published: May 2006

The signals and cellular interactions required for hematopoietic stem-cell commitment to the T lineage are unknown, yet are central to understanding the early stages of normal T-cell development. To study the differentiative capacity of T-cell precursors, we isolated CD4-, CD8-, surface(s) CD3- thymocytes from postnatal human thymuses and determined their capacity to differentiate into lymphoid and nonlymphoid lineages in vitro. We found that CD4-, CD8-, sCD3- thymocytes, which differentiated in the presence of T-cell conditioned medium plus interleukin 2 into T cells expressing the gamma delta receptor for antigen, were capable of differentiating into myeloid or erythroid lineages in the presence of either 5637 bladder carcinoma cell line conditioned medium plus recombinant human erythropoietin or human thymic epithelial cell conditioned medium. Thymic epithelial cell conditioned medium was as effective as 5637 supernatant plus erythropoietin in inducing myeloerythroid differentiation in the CD4-, CD8-, sCD3- thymocytes. Sixty-eight +/- 14% of CD4-, CD8-, sCD3- thymocytes underwent nonlymphoid differentiation within 4 days in culture with 5637 supernatant plus erythropoietin. Twenty-six +/- 4% of freshly isolated CD4-, CD8-, sCD3- cells were CD34+, and clonal granulocyte/macrophage, granulocyte/erythrocyte/monocyte/megakaryocyte, and T-cell progenitors were found in both CD34+ and CD34- subsets of CD4-, CD8-, sCD3- thymocytes. Thus, cells within the human CD4-, CD8-, sCD3- thymocyte subset can give rise to gamma delta+ T cells as well as to cells of myeloerythroid lineages. Moreover, CD34+, CD4-, CD8-, sCD3- cells can give rise to clonal T-cell progenitors as well as to clonal myeloid progenitors.
Herpes simplex virus latent RNA (LAT) is not required for latent infection in the mouse.
Authors: Ho D Y , Mocarski E S
Published: May 2006

During latent infection by herpes simplex virus (HSV), an abundant latency-associated transcript (LAT) that is antisense to a predominant viral alpha gene (ICP0) is found localized in the nucleus of sensory neurons. We disrupted both copies of the LAT gene in the HSV-1 genome by insertion of the Escherichia coli lacZ gene under LAT promoter control. The resulting recombinant virus, RH142, does not express any detectable LAT in either latently or productively infected cells, although beta-galactosidase expression is readily detectable in sensory neurons of latently infected mice. Expression was first detectable 3 days postinoculation and continued at approximately the same level for the entire experimental period (56 days). beta-Galactosidase expression was not detectable at any time during RH142 replication in Vero cells. Thus, the kinetics of expression and cell-type specificity of the LAT gene are distinct from other HSV-1 genes that are expressed during productive growth. When latently infected trigeminal ganglia were explanted, RH142 reactivated from latency with the kinetics and an efficiency indistinguishable from the parental wild-type virus. These studies argue against any possible antisense regulatory mechanism of LAT in the regulation of viral gene expression or any role of LAT-encoded protein during the establishment or maintenance of latency in the mouse.
Characterization of a cDNA clone encoding the visna virus transactivating protein.
Authors: Davis J L , Clements J E
Published: May 2006

The unique pathogenesis of lentiviral infections in humans and ruminant animals may be explained, in part, by the complex mechanisms regulating transcription and translation of their viral genes. This report demonstrates that a visna virus-encoded protein transactivates viral gene expression. A 1.4-kilobase cDNA clone encodes two distinct proteins with apparent molecular masses of 21.5 and 10 kDa. We demonstrate that the 10-kDa species is the visna virus transactivating (Tat) protein; the other species may be analogous to the rev (formerly art or trs) gene product of human immunodeficiency virus.
An odyssey of the green sea turtle: Ascension Island revisited
Authors: Bowen Brian W. , Meylan Anne B. , Avise John C.
Published: May 2006

Green turtles (
Chelonia mydas
) that nest on Ascension Island, in the south-central Atlantic, utilize feeding grounds along the coast of Brazil, more than 2000 km away. To account for the origins of this remarkable migratory behavior, Carr and Coleman [Carr, A. & Coleman, P. J. (1974)
Nature (London)
249, 128-130] proposed a vicariant biogeographic scenario involving plate tectonics and natal homing. Under the Carr-Coleman hypothesis, the ancestors of Ascension Island green turtles nested on islands adjacent to South America in the late Cretaceous, soon after the opening of the equatorial Atlantic Ocean. Over the last 70 million years, these volcanic islands have been displaced from South America by sea-floor spreading, at a rate of about 2 cm/year. A population-specific instinct to migrate to Ascension Island is thus proposed to have evolved gradually over tens of millions of years of genetic isolation. Here we critically test the Carr-Coleman hypothesis by assaying genetic divergence among several widely separated green turtle rookeries. We have found fixed or nearly fixed mitochondrial DNA (mtDNA) restriction site differences between some Atlantic rookeries, suggesting a severe restriction on contemporary gene flow. Data are consistent with a natal homing hypothesis. However, an extremely close similarity in overall mtDNA sequences of surveyed Atlantic green turtles from three rookeries is incompatible with the Carr-Coleman scenario. The colonization of Ascension Island, or at least extensive gene flow into the population, has been evolutionarily recent.
Heterologous expression of a wheat high molecular weight glutenin gene in Escherichia coli.
Authors: Galili G
Published: May 2006

An engineered DNA fragment containing a DNA sequence encoding a wheat high molecular weight glutenin subunit was cloned into a bacterial expression vector that is based on bacteriophage T7 RNA polymerase. The resulting plasmid directed the synthesis of large amounts of the mature form of the wheat high molecular weight glutenin subunit in Escherichia coli. This protein comigrated in SDS/PAGE with a native high molecular weight glutenin subunit extracted from wheat endosperm and cross-reacted with antibodies raised against purified wheat high molecular weight glutenins. The wheat subunit synthesized in E. coli also exhibited pI and solubility characteristics identical to those of native wheat subunits. Moreover, the wheat glutenin subunit produced in E. coli cells self-assembled into oligomers linked by intermolecular disulfide bonds, a process that plays an important role in the assembly of native glutenins during gluten formation. The large amounts of a purified wheat subunit obtained from E. coli will enable investigators to analyze the three-dimensional structure of that protein and to identify protein sequences that affect the bread-making quality of wheat dough.
Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.
Authors: Pang I H , Sternweis P C
Published: May 2006

Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study.
Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells.
Authors: Takeuchi M , Inoue N , Strickland T W , Kubota M , Wada M , Shimizu R , Hoshi S , Kozutsumi H , Takasaki S , Kobata A
Published: May 2006

Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.
Transmembrane domain of the envelope gene of a polycythemia-inducing retrovirus determines erythropoietin-independent growth.
Authors: Chung S W , Wolff L , Ruscetti S K
Published: May 2006

Both the polycythemia-inducing and the anemia-inducing strains of Friend spleen focus-forming virus can induce acute erythroleukemia in susceptible adult mice. However, only cells infected with the polycythemia-inducing strain become erythropoietin-independent for proliferation and differentiation. The sequences responsible for the altered erythropoietin responsiveness have previously been localized to a 678-base-pair EcoRI-Cla I fragment at the 3' end of the envelope gene. This region is now further analyzed by dividing it into two fragments by using the Fok I restriction site. Two recombinants were made by replacing either the 558-base-pair EcoRI-Fok I or the 113-base-pair Fok I-Cla I env gene fragments from the anemia-inducing strain of spleen focus-forming virus with sequences derived from the polycythemia-inducing strain. Our results indicate that the 113-base-pair Fok I-Cla I fragment, which encodes primarily the transmembrane domain of the envelope protein, determines erythropoietin-independent growth.
Pancreatic islet-specific T-cell clones from nonobese diabetic mice.
Authors: Haskins K , Portas M , Bergman B , Lafferty K , Bradley B
Published: May 2006

We have produced a panel of islet-specific T-cell clones from nonobese diabetic (NOD) mice. These clones proliferate and make interleukin 2 in an antigen-specific manner in response to NOD antigen-presenting cells and islet cells. Most of the clones respond to islet-cell antigen from different mouse strains but only in the presence of antigen-presenting cells bearing the class II major histocompatibility complex of the NOD mouse. In vivo, the clones mediate the destruction of islet, but not pituitary, grafts. Furthermore, pancreatic sections from a disease transfer experiment with one of the clones showed a pronounced cellular infiltration and degranulation of islets in nondiabetic (CBA x NOD)F1 recipients.
Hypothesis: microtubules, a key to Alzheimer disease.
Authors: Matsuyama S S , Jarvik L F
Published: May 2006

Alzheimer disease (AD) is a clinicopathologic syndrome of unknown etiology with numerous abnormalities in neuronal and nonneuronal cells. A review of the literature suggests that a common basic intracellular defect may underlie many of the reported abnormalities. We hypothesize impairment of the microtubule (MT) system as one explanation for the pathogenesis of AD. Evidence in support of the hypothesis includes the following: MTs are ubiquitous and vital cell components, unequally distributed, with the highest concentration in the brain; various abnormalities, including the key neuropathologic lesions, can be explained by impairments of the MT system; and experiments utilizing pharmacologic agents known to disrupt MTs have reproduced certain abnormalities observed in AD. The hypothesis provides a framework for systematic investigations of MTs at the cellular and molecular levels as well as the basis for in vivo diagnostic tests for AD.
Molecular cloning of a putative tetrodotoxin-resistant rat heart Na+ channel isoform.
Authors: Rogart R B , Cribbs L L , Muglia L K , Kephart D D , Kaiser M W
Published: May 2006

Voltage-gated Na+ channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na+ channels are blocked by 1-10 microM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity [3H]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire alpha subunit of a cardiac-specific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.
Phytotoxicity of foliar-applied urea
Authors: Krogmeier Michael J. , McCarty Gregory W. , Bremner John M.
Published: May 2006

Recent work in our laboratory showed that the adverse effect of urea fertilizer on seed germination and seedling growth in soil is due to ammonia produced through hydrolysis of urea by soil urease (NH
2
CONH
2
+ H
2
O → 2NH
3
+ CO
2
) and can be eliminated by amending the fertilizer with a small amount of a urease inhibitor such as phenylphosphorodiamidate. Because the leaf-tip necrosis often observed after foliar fertilization of plants with urea is usually attributed to ammonia formed through hydrolysis of urea by plant urease, we studied the possibility that this necrosis could be eliminated or reduced by adding phenylphosphorodiamidate to the urea fertilizer. We found that, although addition of this urease inhibitor to foliar-applied urea increased the urea content and decreased the ammonia content and urease activity of soybean [
Glycine max.
(L.) Merr.] leaves fertilized with urea, it increased the leaf-tip necrosis observed after fertilization. We conclude that this necrosis resulted from accumulation of toxic amounts of urea rather than from formation of toxic amounts of ammonia. This conclusion was supported by our finding that the necrotic areas of soybean leaves treated with urea or with urea and phenylphosphorodiamidate contained much higher concentrations of urea than did the nonnecrotic areas.
Localization of virus-specific and group-specific epitopes of plant potyviruses by systematic immunochemical analysis of overlapping peptide fragments.
Authors: Shukla D D , Tribbick G , Mason T J , Hewish D R , Geysen H M , Ward C W
Published: May 2006

Virus-specific or group-specific antibody probes to potyviruses can be produced by targeting the immune response to the virus-specific, N-terminal region of the capsid protein (29-95 amino acids depending on the virus) or to the conserved core region (216 amino acids) of the capsid protein, respectively. Immunochemical analysis of overlapping, synthetic octapeptides covering the capsid protein of the Johnsongrass strain of Johnsongrass mosaic virus (JGMV-JG) has delineated the peptide sequences recognized by five polyclonal rabbit antisera and two mouse monoclonal antibodies (mAbs). The antibodies characterized were (i) three virus-specific rabbit polyclonal antisera and one virus-specific mouse mAb (1/25) raised against native virus particles, (ii) one polyclonal antiserum raised against trypsin-derived core particles of JGMV-JG, (iii) one group-specific polyclonal antiserum raised against the denatured, truncated coat protein from trypsin-derived core particles of JGMV-JG, and (iv) one group-specific mouse mAb (1/16) raised against native virus particles. The two epitopes seen by mAb 1/25 occurred at residues 18-27 and 43-52 and overlapped with the two major epitopes seen by the virus-specific polyclonal antiserum. The group-specific epitope seen in JGMV-JG by mAb 1/16 was also recognized strongly in potato virus Y, the type member of the potyvirus group. The multiple epitopes seen by the cross-reactive polyclonal antisera were distributed across the entire core region of the coat protein and their relative antibody binding responses varied between JGMV-JG, potato virus Y, and six other distinct potyviruses.
Human 72-kilodalton type IV collagenase forms a complex with a tissue inhibitor of metalloproteases designated TIMP-2.
Authors: Goldberg G I , Marmer B L , Grant G A , Eisen A Z , Wilhelm S , He C S
Published: May 2006

Simian virus 40 (SV40)-transformed human lung fibroblasts secrete both 72-kDa type IV collagenase and a closely related 92-kDa type IV collagenase that was not detected in the parental cell line. The 92-kDa type IV procollagenase purified from these cells exists in a noncovalent complex with the tissue inhibitor of metalloproteases, TIMP. Here we report that the 72-kDa type IV procollagenase purified from HRAS-transformed human bronchial epithelial cells, SV40-transformed lung fibroblasts, and normal skin fibroblasts exists in a stable but noncovalent stoichiometric complex with a 24-kDa inhibitor referred to here as "TIMP-2." TIMP-2 is closely related to TIMP, as demonstrated by comparison of the partial amino acid sequence of this protein to that of TIMP, although it does not cross-react with TIMP-specific antibody. The TIMP-2 inhibitor interacts with the 72-kDa type IV collagenase in preference to the 92-kDa type IV collagenase that forms a complex exclusively with TIMP. The 72-kDa type IV collagenase-TIMP-2 complex can be activated with organomercurials to yield a catalytically competent enzyme. Activation occurs concomitantly with autoproteolytic cleavage of the amino terminus of the protein and does not require dissociation of the complex. Both activity and activation of the complex can be completely inhibited by further addition of stoichiometric quantities of purified TIMP-2 or recombinant TIMP.
Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer.
Authors: Larsen R D , Rajan V P , Ruff M M , Kukowska-Latallo J , Cummings R D , Lowe J B
Published: May 2006

We have developed a genetic approach to isolate cloned cDNA sequences that determine expression of cell surface oligosaccharide structures and their cognate glycosyltransferases. A cDNA library was constructed in a mammalian expression vector by using mRNA from a murine cell line known to express a UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase [(alpha 1-3)GT; EC 2.4.1.151]. This library was transfected into COS-1 cells, which lack expression of (alpha 1-3)GT. Transfected cells containing functional (alpha 1-3)GT cDNAs were detected and isolated with a lectin that recognizes the surface-expressed glycoconjugate product of the (alpha 1-3)GT enzyme. One cloned (alpha 1-3)GT cDNA was rescued from lectin-positive transfected cells. This cDNA contains a single long open reading frame that predicts a 394-amino-acid protein. No significant primary structure similarities were identified between this protein and other known sequences. However, the protein predicts a type II transmembrane topology similar to two other mammalian glycosyltransferases. This topology places the large COOH-terminal domain within the Golgi lumen; this domain was shown to be catalytically active when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. Biochemical analysis confirmed that this enzyme catalyzes a transglycosylation reaction between UDP-Gal and Gal(beta 1-4)GlcNAc to form Gal(alpha 1-3)Gal(beta 1-4)GlcNAc. This cloning approach may be generally applicable to the isolation of cDNAs encoding other mammalian glycosyltransferases.
Transformation stimulates glucose transporter gene expression in the absence of protein kinase C.
Authors: Hiraki Y , Garcia de Herreros A , Birnbaum M J
Published: May 2006

The rat brain glucose transporter (GT) gene is rapidly activated coincident with the initiation of growth in response to oncogenic transformation or the addition of growth factors to quiescent fibroblasts. The latter response has been shown to be mediated by protein kinase C-dependent and-independent pathways. We studied the role of protein kinase C in the transformation-induced activation of the GT gene. Transformation of fibroblasts by either the v-fps or the Ki-ras oncogene rapidly increased the levels of GT mRNA. Either viral oncogene remained capable of stimulating the GT gene after depletion of cellular protein kinase C by prolonged pretreatment of fibroblasts with phorbol 12-myristate 13-acetate. These data indicate that protein kinase C is not required for the rapid activation of gene transcription by oncogenic transformation.
Structure and activity of the sevenless protein: a protein tyrosine kinase receptor required for photoreceptor development in Drosophila.
Authors: Simon M A , Bowtell D D , Rubin G M
Published: May 2006

The sevenless gene encodes a putative protein tyrosine kinase receptor that is required for the proper differentiation of the R7 photoreceptor cells of the Drosophila eye. We have expressed the sevenless protein in Drosophila tissue culture cells and studied its synthesis, processing, and activity. Our results show that the sevenless protein possesses protein tyrosine kinase activity. The protein is first synthesized as a 280-kDa glycoprotein precursor that is subsequently cleaved into 220-kDa amino-terminal and 58-kDa carboxyl-terminal subunits that remain associated by noncovalent interactions. The 220-kDa subunit is glycosylated and contains most of the extracellular portion of the protein, and the 58-kDa subunit is composed of a small portion of the extracellular sequences and the intracellular protein tyrosine kinase domain. This complex is subsequently cleaved into either 49- or 48-kDa carboxyl-terminal fragments with concomitant degradation of the rest of the protein.
Hydrophobic effect in protein folding and other noncovalent processes involving proteins.
Authors: Spolar R S , Ha J H , Record M T
Published: May 2006

Large negative standard heat capacity changes (delta CP degree much less than 0) are the hallmark of processes that remove nonpolar surface from water, including the transfer of nonpolar solutes from water to a nonaqueous phase and the folding, aggregation/association, and ligand-binding reactions of proteins [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. USA 74, 2236-2240]. More recently, Baldwin [Baldwin, R. L. (1986) Proc. Natl. Acad. Sci. USA 83, 8069-8072] proposed that the delta CP degree of protein folding could be used to quantify the contribution of the burial of nonpolar surface (the hydrophobic effect) to the stability of a globular protein. We demonstrate that identical correlations between the delta CP degree and the change in water-accessible nonpolar surface area (delta Anp) are obtained for both the transfer of nonpolar solutes from water to the pure liquid phase and the folding of small globular proteins: delta CP degree/delta Anp = -(0.28 +/- 0.05) (where delta Anp is expressed in A2 and delta CP degree is expressed in cal.mol-1.K-1; 1 cal = 4.184 J). The fact that these correlations are identical validates the proposals by both Sturtevant and Baldwin that the hydrophobic effect is in general the dominant contributor to delta CP degree and provides a straightforward means of estimating the contribution of the hydrophobic driving force (delta Ghyd degree) to the standard free energy change of a noncovalent process characterized by a large negative delta CP degree in the physiological temperature range: delta Ghyd degree congruent to (80 +/- 10)delta CP degree.
Olfactory marker protein gene: its structure and olfactory neuron-specific expression in transgenic mice.
Authors: Danciger E , Mettling C , Vidal M , Morris R , Margolis F
Published: May 2006

Olfactory marker protein (OMP) genomic clones were isolated from a Charon 4A phage lambda rat genomic library. A 16.5-kilobase (kb) fragment of the rat genome containing the gene was isolated and characterized. Sequence analysis of the gene showed the absence of introns and the lack of CAAT and TATA boxes in the 5' flanking region. The transcription initiation site was mapped, and two sites 55 and 58 base pairs upstream of the ATG were observed. The 5' flanking region is rich in G+C residues and contains a G+C-rich motif as well as direct and inverted repeats. Functional OMP regulatory sequences were demonstrated in transgenic mice. An 11-kb chimeric gene was constructed in which the coding region for OMP was replaced with that for Thy-1.1. In Thy-1.2 mice carrying this transgene, Thy-1.1 was expressed solely by olfactory receptor neurons and their axons and terminals in the olfactory bulb.
Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispairs.
Authors: Au K G , Clark S , Miller J H , Modrich P
Published: May 2006

Mutations in the mutY gene of Escherichia coli confer hypermutability reflecting G.C to T.A transversion mutations and result in a deficiency in methyl-independent G-A to G.C mismatch correction. In the present work, the mutY product has been purified to near homogeneity by virtue of its ability to restore G-A to G.C mismatch correction to cell-free extracts of a mutS mutY strain. The 36-kDa protein renders the strand containing the mispaired adenine labile to base-catalyzed cleavage and sensitive to cleavage by several apurinic/apyrimidinic-site endonucleases, with the sites of strand scission by both agents corresponding to the location of the mismatch. These findings indicate that MutY is a DNA glycosylase that hydrolyzes the glycosyl bond linking the mis-paired adenine to deoxyribose. MutY, a 5'-apurinic/apyrimidinic-site endonuclease, DNA polymerase I, and DNA ligase are sufficient to reconstitute MutY-dependent G-A to G.C repair in vitro.
Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease.
Authors: Wolff J A , Fisher L J , Xu L , Jinnah H A , Langlais P J , Iuvone P M , O'Malley K L , Rosenberg M B , Shimohama S , Friedmann T
Published: May 2006

Rat fibroblasts were infected with a retroviral vector containing the cDNA for rat tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. A TH-positive clone was identified by biochemical assay and immunohistochemical staining. When supplemented in vitro with pterin cofactors required for TH activity, these cells produced L-dopa and released it into the cell culture medium. Uninfected control cells and fibroblasts infected with the TH vector were grafted separately to the caudate of rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. Only grafts containing TH-expressing fibroblasts were found to reduce rotational asymmetry. These results have general implications for the application of gene therapy to human neurological disease and specific implications for Parkinson disease.